Dichloromethylbenzene

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 October 2018-14 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The objective of this study was to evaluate the pre- and post-natal effects of Dichlorotoluene Mixture, an industrial chemical, when administered orally, by gavage, to Han Wistar rats. The evaluation included assessment of the integrity and performance of the adult male and female reproductive tract, and systemic toxicity in pregnant and lactating females and in young and adult offspring. The study was requested by the ECHA Decision number TPE D 2114394001-60-01/F.
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies and as foreseen in the test guideline. The RccHan™;WIST was used because of the historical control data available at this laboratory.
The oral gavage route of administration was chosen as it is a possible route of human exposure, as requested by ECHA Decision number TPE D 2114394001-60-01/F.

Test material

Test material information

Constituent 1

Reference substance name:

Dichloromethylbenzene

EC Number:

249-854-8

EC Name:

Dichloromethylbenzene

Cas Number:

29797-40-8

Molecular formula:

C7H6Cl2

IUPAC Name:

(dichloromethyl)benzene

Test material form:

liquid

Details on test material:

Identification: Dichloromethylbenzene
Appearance: clear colourless liquid
Test substance storage: Refrigeratoted (2-8°C) under nitrogen in the dark
Molecular formula: C7H6Cl2
Molecular weight: 161.0
Volatile: vapour pressure: 65.3 Pa at 298 K
Specific gravity/density: 1.256 kg/L (20˚C)
Purity (GC-FID): 100 %
Batch: CHW 162018

Specific details on test material used for the study:

Test item: Dichlorotoluene Mixture
Test item identity (including alternative names): Dichloromethylbenzene
Molecular formula C7H6Cl2
CAS name Benzene, dichloromethyl-
CAS number: 29797-40-8
Intended use: Industrial chemical
Appearance: Clear colorless liquid
Storage conditions: Refrigerated (2-8°C) under Nitrogen in the dark
Supplier: Sponsor
Batch number: CHW162018
Expiry date: 18 April 2020
Purity (GC-FID) 100%

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Strain/Species RccHan™;WIST rat.
Supplied as 54 litters of identified litter mates.
27 litters of four male siblings and 27 litters of four female siblings; no male/female sibling relationships
Duration of acclimatization for F0 animals Six days before commencement of treatment.
Age of the F0 animals at the start of treatment 28 to 34 days old.
Weight range of the F0 animals at the start of treatment Males: 64 to 100 g. Females: 63 to 99 g.
Age of the F0 animals at the start of treatment 28 to 34 days old.
Supplier Envigo RMS (UK).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal facility
Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering lactation and maturation periods.
Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding Solid bottom cages contained softwood based bark-free fiber bedding, sterilized by autoclaving, which was changed at appropriate intervals each week.
Diet Supply
Diet SDS VRF1 Certified pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
The phytoestrogen content of the diet was controlled (VRF1 has 144 ppm bioavailable Total Genistein Equivalents).
Availability Non-restricted (removed overnight before blood sampling for hematology, blood chemistry, urinalysis and biomarkers investigations (except F1 offspring on Days 4 or 22 of age).
Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at the same time that cages were changed.
Availability Non-restricted (except during urine collection).
Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Environmental Enrichment
Aspen wood-based products A soft white untreated wood block; provided to each cage throughout the study (except during pairing and overnight for urine collection) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing and lactation and overnight for urine collection) and replaced at the same time as the cages.
For F0 females, shelters were returned on Day 21 of lactation after weaning of offspring.
Paper shavings From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen wood-based products.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

Group Treatment Dose(mg/kg bw/day)1,2 Nominal concentration(mg/mL) Volume dose(mL/kg)
1 Vehicle 0 0 5
2 Dichlorotoluene Mixture 50 10 5
3 Dichlorotoluene Mixture 150 30 5
4 Dichlorotoluene Mixture 500# 100 5
4 Dichlorotoluene Mixture 300$ 60 5
1 Dose (mg/kg/day) administered to F0 in Weeks 1-10#
2 Dose (mg/kg/day) administered from F0 Week 11$
Correction factor None.
Vehicle Polyethylene glycol 400 (specific gravity 1.125).
Method of preparation The required amount of test item was weighed into a suitable container. Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the test item and was magnetically stirred until uniformly mixed. A further amount of vehicle was added to make up to the required volume and further mixing, using a magnetic stirrer, was performed until the formulation was homogeneous. The remaining concentrations were then formulated in ascending order of concentration.
Frequency of preparation Weekly.
Storage of formulation Refrigerated (2 to 8 C).
Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Details on mating procedure:

Pairing commenced After ten weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups (sibling pairing was not permitted).
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray.
Vaginal smear - examined for the presence of spermatozoa and the stage of the estrous cycle.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis
Stability and homogeneity Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
In that study, stability was demonstrated for one day (24 hours) at room temperature (15 to 25°C) and 15 days refrigerated (2 to 8°C).
Achieved concentration Samples of each formulation prepared for administration in Week 1 of F0 and F1 generation, Week 11 of F0 generation and the last week of F1 generation of treatment were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

F0 animals For ten weeks before pairing until termination after litters were weaned.
F1 animals From weaning until termination of respective cohort.
Oral gavage - Direct treatment of F1 offspring commences at weaning (Day 21 of age); although direct treatment started at weaning, all offspring had potential for exposure in utero and via the milk during lactation.
Cohort 1A : General toxicity and pathology of the tissues of the
male and female reproductive systems.
Treated from weaning to 13 weeks of age
Cohort 1B : Spare Cohort
Treated from weaning to approximately 14 weeks of age.

Frequency of treatment:

Once daily at approximately the same time each day. A female was not dosed if parturition was in progress at the scheduled time of administration.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control (Polyethylene Glycol 400)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Dichlorotoluene mixture -Low dose

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Dichlorotoluene mixture-mid dose

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Dichlorotoluene mixture - High dose which was reduced to 300mg/kg/day after 11 weeks of treatment

No. of animals per sex per dose:

F0 Generation

Group Treatment Dose (mg/kg/day) Number of animals
Male Female
1 Control 0 25 25
2 Dichlorotoluene Mixture 50 25 25
3 Dichlorotoluene Mixture 150 25 25
4 Dichlorotoluene Mixture# 500 25 25
4 Dichlorotoluene Mixture$ 300
# Dose (mg/kg/day) administered to F0 in Weeks 1-10
$ Dose (mg/kg/day) administered from F0 Week 11
F1 Generation
Cohort Group Treatment Dose (mg/kg/day) Number of animals
Male Female
A 1 Control 0 20 20
2 Dichlorotoluene Mixture 50 20 20
3 Dichlorotoluene Mixture 150 20 20
4 Dichlorotoluene Mixture 300 20 20

B 1 Control 0 20 20
2 Dichlorotoluene Mixture 50 20 20
3 Dichlorotoluene Mixture 150 20 20
4 Dichlorotoluene Mixture 300 20 20

Control animals:

yes, concurrent vehicle

Details on study design:

Doses selected for this study were based on the findings in the preliminary study (Envigo Study Number: YB58QG). In that study oral gavage administration of Dichlorotoluene Mixture at 250, 500 or 1000/750 mg/kg bw/day was related to distinct toxicity including mortality at the highest dose (three males/one female). In F0 males hepatocellular hypertrophy and accumulation of hyaline droplets in kidneys were seen starting at the low dose of 250 mg/kg bw/day. At the high dose reduced live birth index and low birth weight, with reduced pup growth to Day 35 were as well observed. Therefore 500 mg/kg bw/day was selected as the highest dose and the dose levels of 150 mg/kg bw/day and 50 mg/kg bw/day as suitable lower dosages.Treatment at 500 mg/kg/day was not tolerated by a small number of animals. Four animals died or were killed for welfare reasons within the ten week pre pairing treatment period following brief periods of respiratory distress and two had blocked nasal turbinates. Treatment at 500 mg/kg/day was reduced to 300 mg/kg/day from Week 11; Day 1 (day of pairing).

Examinations

Parental animals: Observations and examinations:

Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary
Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule
F0 males Week 1 - daily
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onwards - once each week
F0 females Week 1 - daily
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onwards - once each week
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 7, 14 and 21
F1 generation
Day 21 of age to nominal Day 28 (formal commencement of F1)
Week 1 - daily
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onwards - once each week
Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing
As late as possible in the working day
Clinical Signs
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
F0 males and selected F1 generation Once each week
F0 females Once each week until paired for mating
Gestation phase - Days 0, 5, 12, 18 and 20
Lactation phase - Days 1, 7, 14 and 21
A detailed physical examination was performed at nominally the same time of day on each occasion by an observer. After removal from the home cage, animals were assessed for physical condition and behavior during handling. Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior. Any deviation from normal was recorded with respect to nature, and, where appropriate, degree of severity.
Body Weight
The weight of animals was recorded as follows:
F0 males Before dosing on the day that treatment commenced (Week 0) and weekly thereafter
On the day of necropsy
F0 females Before dosing on the day that treatment commenced (Week 0) and weekly until paired for mating
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating
Days 1, 4, 7, 14, 21 and 28 post-partum
On the day of necropsy
F1 generation selected animals From nominal four weeks of age, twice during Week 1 of the F1 generation and weekly thereafter
On the day of necropsy
Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 males Each week until paired for mating
F0 females Each week until paired for mating
Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17, 18-19
after mating
Days 1-3, 4-6, 7-13 and 14-20 of lactation
F1 generation selected animals From nominal four weeks of age, twice during Week 1 of the F1 generation and weekly thereafter
From these records the mean consumption per animal (g/animal/day) or (g/animal/week) was calculated for each phase.
Mating Procedure (F0 Generation)
Pairing commenced After ten weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups (sibling pairing was not permitted).
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray.
Vaginal smear - examined for the presence of spermatozoa and the stage of the estrous cycle.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.
Parturition Observations and Gestation Length (F0 Generation)
Duration of gestation Time that elapsed between mating and commencement of parturition.
Parturition observations From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.
Biomarkers (TSH and T4)
Blood samples were collected from animals at the following timepoints
Occasion Generation Animals
Termination F0 adults Ten male and ten female animals per group
F1 offspring Σ Ten litters per group - pooled litter sample Day 4 of age
Ten male and ten females per group on Day 22 of age from as many litters as possible
F1 adults - Cohort A Ten male and ten female animals per group
Σ Restricted to T4 only
Conditions Adults: Following overnight deprivation of food.
Offspring: No overnight deprivation of food.
Sample site Adults: Sublingual vein
Offspring Day 4 of age: Decapitation
Offspring Day 22 of age: Orbital sinus
Anesthetic Adults and offspring on Day 22 of age: Isoflurane.
Offspring Day 4 of age: not required.
Anticoagulant None.
Blood tube Greiner Minicollect tubes with clotting activator and gel separator.
Sample volume Adults and offspring Day 22 of age: 1 mL.
Offspring Day 4 of age: maximum possible.
Treatment of samples Samples were kept at ambient temperature (15-25°C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000 g for ten minutes at 4°C.
Number of serum aliquots/tubes Adults and offspring Day 22 of age: two aliquots (tubes) per animal (one for T4 and one for TSH).
Offspring Day 4 of age: one aliquot (tube) - T4 only.
Samples were transferred to appropriately labelled polypropylene “cryo” (Standard Covance) tubes, using plastic disposable pipettes.

Aliquot volumes Adults and offspring at Day 22 of age:
Aliquot 1: T4 - 0.2 mL of serum
Aliquot 2: TSH - residual serum
Offspring Day 4 of age: single aliquot for T4 analysis, all available collected.
Final storage conditions Deep frozen (-60°C to -90ºC), pending analysis.
Fate of plasma samples Dispatched to the Department of Bioanalysis/Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance on dry ice.

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food. Sampling for Cohort A was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Ten male and ten female animals per group
F1 Cohort A Ten male and ten female animals per group

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

* Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. A manual count of the differential white blood cell parameters was performed, when counts of basophils, eosinophils or large unstained cells was higher than 5%.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent
Blood Chemistry
Blood samples were collected after overnight withdrawal of food. Sampling for Cohort A was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Ten male and ten female animals per group
F1 Cohort A Ten male and ten female animals per group

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Direct bilirubin (BILD)
Indirect bilirubin (INDC)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.
Urinalysis - F1 Cohort 1A Generation
Urine samples were collected after overnight withdrawal of food and water at the following occasion:
Occasion Generation Animals
Termination F1 Cohort A Ten males and ten females animals per group

The individual samples were examined for the following characteristics:
Using manual methods:
Appearance (App) - by visual assessment
Volume (Vol) - using a measuring cylinder
pH - using a pH meter
Specific gravity (SG) - by direct refractometry using a SG meter

Using Multistix reagent strips interpreted using the Clinitek®500 instrument (semi-quantitative method):
Glucose (Gluc)
Ketones (Keto)
Bile pigments (Bili)
Blood pigments (UBld)

Using a Cobas 6000 Analyzer (quantitative automated method):
Protein - total (T-Prot) and concentration (Prot)
Sodium - total (T-Na) and concentration (U-Na)
Potassium - total (T-K) and concentration (U-K)
Chloride - total (T-Cl) and concentration (U-Cl)

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.
Epithelial cells (Epi)
Leucocytes (WBC)
Erythrocytes (RBC)
Casts
Other abnormal components (A)

The slide was also examined for abnormalities in spermatozoa and crystals.
If there was insufficient sample to analyze all the parameters listed above, the following priority list applied:
Clarity
Colour
Volume
pH
Specific gravity
Clinitek list
P-mod list
Microscopy
Sexual Maturation (Selected F1 Generation Only)
Males Sexual maturation was assessed by daily examination from Day 35 of age until balano-preputial separation occurred. Body weight was recorded on the day of completion of separation.
Females Sexual maturation was assessed by daily examination from Day 25 of age until vaginal opening occurred. Body weight was recorded on the day of vaginal opening.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears Daily for 15 days before pairing, using cotton swabs moistened with saline.
Wet smears After pairing until evidence of mating confirmed.
For four days before scheduled termination (nominally Days 25 to 28 post-partum) daily vaginal smears were taken and used to determine the stage of the estrous cycle at termination.
Estrous Cycle Monitoring (F1 Generation - Cohort A)
Dry smears Smears were taken for two weeks from approximately Day 75 of age, using cotton swabs moistened with saline.
Wet smears (using pipette lavage) Following onset of vaginal opening until the first cornified (estrus) smear was recorded.
For at least three days prior to the start of the necropsy phase and on the day of termination.
Estrous Cycle Monitoring (F1 Generation - Cohort B)
Wet smear (using pipette lavage) For at least three days prior to the start of the necropsy phase and on the day of termination.

Sperm parameters (parental animals):

Sperm Analysis (F0 and F1 Cohort A males)
Immediately after scheduled sacrifice of each male and collection of blood, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.
The following tests were performed:
Sperm motility - all groups A sample of sperm was expressed from the left vas deferens (the right vas deferens only was used for Group 1 male 7 and Group 3 male 60 for F0 generation) into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and at least 200 sperm per animal analyzed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyzer (CASA).
Sperm morphology - Groups 1 and 4 A 200 µL aliquot of the sperm/medium mixture (described above) was diluted with 800 µL of 10% neutral buffered formalin. After staining with nigrosine and eosin an air dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, where possible (Group 1 Male 402 unable to assess 200 sperm).
Groups 2 and 3 Fixed samples retained for possible future assessment.
Sperm count -
all groups The left cauda epididymis of each male was weighed and then the tunica was removed. The portion obtained was weighed then frozen. Prior to analysis the cauda epididymis portions were allowed to thaw then homogenized for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.
Group 2 and 3 Retained frozen for possible future assessment.
Homogenization-resistant spermatids count -
all groups After removal of the tunica, the left testis of each male (the right testis only was used for Group 1 male 7 and Group 3 male 60 for F0 generation) was frozen. Prior to analysis, the testes were allowed to thaw then homogenized for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenization-resistant spermatid count using CASA.
Group 2 and 3 Retained frozen for possible future assessment.

Litter observations:

Records Made During Littering Phase (F0 Generation)
The records maintained were as follows:
Clinical observations All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter.
On Day 1 of age all offspring received a qualitative assessment of body temperature, state of activity and reaction to handling.
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age.
On Day 4 of age, litters containing more than eight offspring were reduced to eight by random culling (no formal method) by the operator, leaving, whenever possible, four male and four female offspring in each litter; culled offspring were subjected to macroscopic examination.
Sex ratio of each litter Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
Individual offspring body weights Recorded on Days 1, 4 (before culling), 7, 14, 21, 23, 25, 27α and 29α of age.
α Only applicable before formal commencement of the F1 generation at nominal four weeks of age (Day 28 of age ± 2 days).
Weaning of offspring The dam was removed from the litter cage and offspring were weaned on Day 21 of age.
Ano-genital distance Day 1 of age.
Nipple/areolae count Day 13 of age - male offspring only.

Postmortem examinations (parental animals):

Time of Necropsy
F0 males After weaning of the F1 animals, after confirmation that no further mating required.
F0 females failing to produce a viable litter Terminated with first cohort of females with live litters.
F0 females Day 28 post-partum.
Unselected offspring On Day 4 and Day 22 of age.
Unselected F1 spares, following one week of treatment to the F1 generation.
F1 adults (Cohort A) Approximately 13 weeks of age.
F1 adults (Cohort B) Approximately 14 weeks of age.
Method of Kill
Offspring before Day 14 of age Animals less than 14 days of age: Intraperitoneal injection of sodium pentobarbitone.
Offspring from Day 14 of age and older Animals 14 days and older: Carbon dioxide asphyxiation with subsequent exsanguination.
Sequence To allow satisfactory inter-group comparison.
Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
F0 Females
The following were recorded:
Each uterine horn Number of implantation sites was counted and confirmed, using Salewski stain, if none were visible at visual inspection.
Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified in the relevant pathology procedures table. Requisite organs were weighed for animals killed at scheduled intervals.
For F1 unselected offspring, ten male and ten female per group were selected at random from litters for organ weights.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed in attached pathology tables
Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List All adult animals killed prematurely.
All F0 and F1 Cohort A terminal adult animals of Groups 1 and 4 at scheduled sacrifice.
Abnormalities only All F0 and F1 Cohort A terminal adult males and females of Groups 2 and 3 at scheduled sacrifice. All animals in F1 Cohort B.
Block stage only Reproductive organs for all F1 Cohort B terminal adult animals.
Routine staining Sections were stained with hematoxylin and eosin.
3.7.9 Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Premature deaths
Scheduled kill All F0 and F1 animals from all groups. All specified in table attached.
F0 animals in Groups 1 and 4 All specified in table attached .
All males of Groups 2 and 3. Kidney and liver and abnormalities only
F0 females in Groups 2 and 3 Abnormalities only.
F0 animals in Groups 2 and 3 with suspect fertility Reproductive organs only.
F1 - Cohort A animals in Groups 1 and 4 All specified in attached table.
F1 - Cohort A males in Groups 2 and 3 Kidney and liver and abnormalities only.
F1 - Cohort A females in Groups 2 and 3 Abnormalities only.
F1 - Cohort B in all animals Abnormalities only.

For the assessment of the testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made for F0 females. For F1 Cohort A females qualitative examination of ovarian tissue, a quantitative assessment was made of the primordial follicle and small growing follicle populations as well as corpora lutea population (differential follicular enumeration). For this, five sections were cut at about 100m intervals from the inner third of each ovary and the primordial follicles and small growing follicle populations counted.
The stage of vaginal estrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands).
All other findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
Immunophenotyping of spleen leucocytes (F1 - Cohort A)
Ten males and ten females per group (one male or one female from each litter, all litters were represented by at least one offspring) were selected for immunophenotyping. After the spleen was weighed a 3-5 mm mid transverse section was removed and retained for histopathological examination. The remaining spleen was weighed and then placed into a vial of Hank’s Balanced Salt Solution (HBSS) and held on wet ice until processing for analysis. Samples were dispatched to Department of Bioanalysis, Biomarkers and Clinical Sciences, Covance.
The analyses were performed by the Department of Bioanalysis, Biomarkers and Clinical Sciences, Covance

Postmortem examinations (offspring):

F1 Offspring
Offspring were subject to a complete macroscopic examination. Additionally, the following procedures were applicable:
Premature deaths (before weaning) Missing offspring and any grossly autolyzed or grossly cannibalized could not be examined. All other remaining offspring dying before weaning were examined; where possible the examination also included an assessment for the presence of milk in the stomach.
The organs weighed and tissue samples fixed are detailed in attached pathology tables

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. The similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods
Vaginal opening to first estrus (F1 Cohort A)
Hematology
Blood chemistry
Urinalysis
Sexual maturation, age and body weight at completion
Organ weights, absolute and relative to body weight
Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index, live birth index and viability index), for before cull study periods
Ano-genital distance, adjusted for Day 1 pup body weight
clinical pathology data
live birth and viability indices
vaginal opening to first estrus
litter average ano genital distance data

Reproductive indices:

Mating Performance and Fertility (F0 Females)
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = Number of animals mating x 100
Animals paired

Conception rate (%) = Number of animals achieving pregnancy x 100
Animals mated

Fertility index (%) = Number of animals achieving pregnancy x 100
Animals paired

Gestation Length and Index (F0)
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = Number of live litters born x 100
Number pregnant

Offspring viability indices:

Survival Indices
The following were calculated for each litter:
Post-implantation survival index (%) = Total number of offspring born x 100
Total number of uterine implantation sites

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = Number of live offspring on Day 1 after littering x 100
Total number of offspring born

Viability index (%) = Number of live offspring on Day 4 before culling x 100
Number live offspring on Day 1 after littering

Lactation index (%) = Number of live offspring on Day 21 after littering x 100
Number of live offspring on Day 4 after culling

Group mean values were calculated from individual litter values.
Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after culling) and 21 of age.
Percentage males = Number of males in litter x 100
Total number of offspring in litter

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs and observations attributed to dosing predominantly affected respiration. The signs were short-lived and the majority were terminal/lethal, as indicated by the few animals with respiratory signs recorded at the weekly clinical observation investigations. Very few animals seen with these signs survived.
Incidences of salivation, post salivation staining and respiratory signs (irregular, labored, fast/slow breathing, rales and sneezing) were evident at 50, 150 or 500/300 mg/kg/day and showed strong correlation to dose.

Mortality:

mortality observed, treatment-related

Description (incidence):

In the F0 generation, six animals were killed for welfare reasons and two animals were found dead. Four females that failed to litter were killed on Day 25 after mating.
One female (No. 257) receiving 150 mg/kg/day showed labored and noisy respiration on Day 10 of gestation and was killed for welfare reasons. Macroscopic examination revealed gas in the stomach, jejunum, duodenum, ileum, cecum and rectum contained gas.
One female (No. 278) receiving 500 mg/kg/day was underactive with pallor, rales and irregular respiration on Day 63 and was killed for welfare reasons. Macroscopic examination revealed gas in the jejunum, ileum, colon and caecum and pale areas on the liver.
One female (No. 281) receiving 500 mg/kg/day was found dead on Day 69. The animal had irregular respiration and rales on Day 68. Macroscopic examination revealed gas in the stomach, jejunum and ileum and blocked nasal turbinates that were considered to restrict breathing.
One female (No. 289) receiving 500 mg/kg/day was euthanized for welfare reasons on Day 17, with labored respiration and rales and hunched posture and pallor. Macroscopic examination revealed gas in the stomach, jejunum, ileum and caecum and blocked nasal turbinates that were considered to restrict breathing.
Microscopic examination of these animals revealed findings in some tissues such as lungs, liver, thymus, etc. The major factor contributory to death for these animals was considered to be general poor clinical condition except for No. 281 which was undetermined.
One female (No. 292) receiving 500 mg/kg/day was euthanized for welfare reasons on Day 30 with labored respiration and rales; however, macroscopic examination revealed no findings in any tissues. Microscopic examination revealed findings in the lungs, adrenals and thymus.
One male (No. 83) receiving 500/300 mg/kg/day had labored respiration and rales on Day 84 and was euthanized for welfare reasons. Macroscopic examination revealed dark areas in the lungs. Microscopic examination revealed findings in the liver, kidneys and lungs.
The major factor contributory to death of these animals was considered to be general poor clinical condition.
One male (No. 95) receiving 500/300 mg/kg/day was found with a cut of the skin on the lower ventral surface on the day after successful mating on Day 73 and was euthanized for welfare reasons. Macroscopic examination revealed dark depressions of the skin. At microscopic examination, test item-related findings were seen in the liver and kidneys. Histopathologic findings were also seen in the skin and heart. The major factor contributory to death was considered to be skin lesions.
Male No. 34 receiving 50 mg/kg/day was found dead on Day 99. Macroscopic examination revealed perforated esophagus and abnormal contents of the thoracic cavity. Microscopic findings were seen in the liver and lungs, and major factor contributory to death was considered to be a dosing error.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Overall body weight gain in un-mated females and females during gestation and lactation were considered to be unaffected by treatment. Body weight gain of females receiving 150 mg/kg/day was marginally high, when compared with Control, during gestation (110%); however, as there was no effect on body weight gain at 300 mg/kg/day, it was considered not to be toxicologically significant.
Overall body weight gain in males was unaffected, during 18 weeks of treatment, at 50, 150 or 500/300 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Overall food consumption in un-mated females and females during gestation and lactation was considered to be unaffected by treatment at 50, 150 or 500/300 mg/kg/day.
Food consumption appeared marginally high during Days 10 to 12 of gestation at 50, 150 or 300 mg/kg/day (range 119% to 125% of Control); however, this was attributed to incidental transient low food intake in the Control. Food consumption was statistically significantly low in females receiving 300 mg/kg/day during Days 7-14 of lactation (94%); however the difference from Control was marginal and considered not to be biologically relevant or toxicologically significant.
Food consumption in males, during the 10 week pre-pairing period, was unaffected by treatment at 50, 150 or 500 mg/kg/day.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

It is considered that the hematological examination, following 18 weeks of treatment to males treated at 50, 150 or 500/300 mg/kg/day, revealed no toxicologically significant differences from Control. All differences lacked relationship to dose and were therefore attributed to normal biological variation. These included, high counts of lymphocytes and large unstained cells at 150 or 500/300 (133% or 119% and 300% or 200% of Control, respectively) that contributed to the high white cell count at these doses (139% or 117% of Control, respectively).
The hematological examination in females on Day 28 of lactation, following 17 weeks of treatment at 50, 150 or 500/300 mg/kg/day, revealed no toxicologically significant differences from Control.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

When compared with Control, the biochemical examination of the blood plasma in males in Week 18 and females in Week 17 (on Day 28 of lactation), at 50, 150 or 500/300 mg/kg/day revealed no toxicologically significant differences from Control. All differences were minor and lacked dose relationship and were therefore attributed to normal biological variation.
Minor variations in electrolyte concentrations were evident in males and females, without dose relationship; namely, potassium was high, in males and females receiving 50, 150 or 500/300 mg/kg/day (107%, 111% or 108% and 114%, 103% or 110% of Control, respectively) and phosphorus was high in males only at the same doses (109%, 123% or 107% of Control, respectively). Albumin concentration was marginally high, when compared with Control, in males only at 500/300 mg/kg/day (105%).

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment Related Findings
Changes related to treatment with Dichlorotoluene Mixture were seen in the liver and kidneys of males. No test item-related findings were seen in females.
Liver
Centrilobular hypertrophy was seen in males given 50, 150 or 500/300 mg/kg/day.
Summary of treatment related findings in the liver for F0 males
Group/sex 1M 2M 3M 4M
Dose (mg/kg/day) 0 50 150 500/300
Hypertrophy, Centrilobular
Minimal 0 2 6 6
Slight 0 0 1 10
Total 0 2 7 16
Number of tissues examined 25 24c 25 23b
b Two males (No’s 83 and 95) were killed for welfare reasons.
c One male (No. 34) was found dead.
Kidneys
Increased incidence and severity of hyaline droplet accumulation accompanied by basophilic tubules and/or granular casts was seen in males given 50, 150 or 500/300 mg/kg/day.
Summary of treatment related findings in the kidneys for F0 males
Group/sex 1M 2M 3M 4M
Dose (mg/kg/day) 0 50 150 500/300
Accumulation, Hyaline Droplets
Minimal 1 5 11 1
Slight 0 0 2 11
Moderate 0 0 0 11
Total 1 5 13 23
Basophilia, Tubular
Minimal 1 3 7 9
Slight 0 0 1 9
Total 1 3 8 18
Cast(s), Granular
Minimal 0 0 6 9
Slight 0 0 1 7
Total 0 0 7 16
Number of tissues 25 24c 25 23b
examined
b Two males (No’s 83 and 95) were killed for welfare reasons.
c One male (No. 34) was found dead.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

All other microscopic findings were considered to be incidental and unrelated to the test item.
Seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted. The qualitative examination of the ovary with follicle and corpora lutea counts and staging of the estrus cycle revealed no abnormality.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Pre-pairing estrous cycles were considered to be unaffected at 50, 150 or 500 mg/kg/day.
One female receiving 500 mg/kg/day had an irregular estrous cycle; however as this animal mated within four days of pairing with a male, it was considered not to be toxicologically significant.
All females at 50, 150 or 500/300 mg/kg/day showed estrus, following weaning of litters and before termination (Days 25-28 of lactation)

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

When compared with Control, it was considered that there was no adverse effect on sperm motility, morphology or cauda epididymal sperm following treatment for 18 weeks at 500/300 mg/kg/day.
Testicular spermatid concentration was marginally low at 500/300 mg/kg/day (92% of Control), but was within the Historical Control Data range (HCD).

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, pre-coital interval and fertility were unaffected by treatment at 50, 150 or 500/300 mg/kg/day.
Gestation length and gestation index were unaffected by treatment at 50, 150 or 300 mg/kg/day.
Litter Size, Sex Ratio and Survival Indices
The number of implantations was marginally high, when compared with Control, at 150 or 500/300 mg/kg/day (119% or 106%, respectively), but failed to show relationship to treatment and toxicological significance was not inferred.
Litter size at Day 1 and survival indices to Day 21 of age were unaffected by maternal treatment at 50, 150 or 500/300 mg/kg/day.
The ratio of males to females was unaffected by treatment at 50, 150 or 500/300 mg/kg/day.
Offspring Body Weight
Following maternal treatment and when compared with Control, the body weights of male and female offspring were marginally low (combined; 93%, 89% or 91%, respectively) at 50, 150 or 500/300 mg/kg/day on Day 1 of age, but failed to show relationship to dose. The body weight of Control offspring was higher than, and body weights at 50, 150 or 500/300 mg/kg/day were lower than the HCD range (males 6.4-6.6 g; females 6.1-6.4 g; 7 studies November 15-May 2019)). Subsequent body weight gain to Day 21 of age was marginally lower than Control at all doses, but the differences only attained statistical significance for females at 300 mg/kg/day (94%). It is noted that the mean body weight of the Control exceeded the HCD range (45.7-49.6 g; 7 studies November 15-May 2019).

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Remarks on result:

other: Not evidence of systemic toxicity at high dose

Remarks:

based on Irritancy of test item

Target system / organ toxicity (P0)

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs and observations attributed to dosing predominantly affected respiration. The signs were short-lived and the majority were terminal/lethal, as indicated by the few animals with respiratory signs recorded at the weekly clinical observation investigations. Very few animals seen with these signs survived.
Irregular breathing and rales were evident for the duration of study (Weeks 1-11) in several males and females that received 150 and 300 mg/kg/day. Three separate males receiving 300 mg/kg/day additionally had laboured breathing from Weeks 2-4, one of which also had rapid breathing.
Persistent piloerection was evident in one male receiving 50 mg/kg/day during Weeks 2-5 and another receiving 300 mg/kg/day between Weeks 2-9. Salivation occurred in several males and females that received 150 and 300 mg/kg/day from Weeks 2-7.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

F1 Generation, Cohort A
One female (No. 658) dosed with 150 mg/kg/day was euthanised for welfare reasons on Day 20. The animal had showed gasping, irregular breathing and rales.
One female (No. 663) receiving 300 mg/kg/day was euthanised for welfare reasons on Day 18. The animal had irregular and labored respiration and rales.
One female (No. 672) receiving 300 mg/kg/day was euthanised for welfare reasons on Day 12. The animal showed gasping, irregular and laboured respiration and piloerection and had a firm area of the lower ventral surface.
One female (No. 679) that received 300 mg/kg/day was euthanised for welfare reasons on Day 60. The animal had decreased activity, rales and irregular, laboured and noisy respiration.
Macroscopic examination of these animals revealed gaseous abnormal contents and/or distension of the gastro-intestinal tract. Additionally, blocked nasal turbinates which were unable to flush were seen in No’s. 672 and 679. At microscopic examination, no finding was seen in No. 679 while findings were seen in some tissues such as lungs, liver, spleen, etc., of the other females. The major factor contributory to death was considered to be general poor clinical condition for all animals, except for No. 663 which had a breathing impairment.
One male (No. 452) receiving 150 mg/kg/day was euthanised for welfare reasons on Day 13. The animal had rales, decreased activity, dull eyes and an abnormal tilt of the head and the animal vocalised when handled.
One male (No. 468) receiving 300 mg/kg/day was euthanised for welfare reasons on Day 18. The animal had irregular and laboured respiration and rales.
One female (No. 669) treated with 300 mg/kg/day was euthanised for welfare reasons on Day 40. The animal was found to have persistent piloerection and irregular and laboured breathing with rales and pallor to its body.
At macroscopic examination, no gross abnormality was seen in No’s. 452 and 669, and No. 468 had blocked nasal turbinates which were unable to flush. Microscopic examination revealed findings in some tissues such as liver, spleen, sternum, femur, etc. The major factor contributory to death was considered as breathing impairment for all animals except for No. 452 which was considered as general poor clinical condition.
One male (No. 445) receiving 150 mg/kg/day was found dead on Day 14. Macroscopic examination revealed a perforated esophagus and abnormal contents of the thoracic cavity. At microscopic examination, findings were seen in the spleen, sternum, femur, epididymides, prostate and seminal vesicles. The major factor contributory to death was considered to be a dosing error.
One female (No. 750) dosed with 300 mg/kg/day was found dead with red staining (blood) around the nose and muzzle on Day 9. Macroscopic examination revealed gaseous abnormal contents and/or distension in the stomach and a blocked left nasal turbinate which was unable to flush. At microscopic examination, findings were seen in the lungs, spleen, sternum, femur, trachea and nose, and the major factor contributory to death was considered to be respiratory lesions.
One male (No. 521) receiving 150 mg/kg/day was euthanised for welfare reasons on Day 43. The animal showed irregular and labored respiration and rales.
One female (No. 719) receiving 50 mg/kg/day was euthanised for welfare reasons on Day 15. The animal showed gasping respiration, underactive behavior and whole body cyanosis (blue).
At macroscopic examination, no gross abnormality was seen in No. 521. No. 719 had perforated and dark areas of the esophagus, a pale and small spleen, a distended stomach and, pale adhesions and abnormal contents of the thoracic cavity. Microscopic examination revealed findings in some tissues such as lungs, liver, kidneys, etc. The major factor contributory to death was considered to be a dosing error for No. 719 and general poor clinical condition for No. 521.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Following maternal treatment and when compared with Control, the body weights of male and female offspring were marginally low (combined; 93%, 89% or 91%, respectively) at 50, 150 or 500/300 mg/kg/day on Day 1 of age, but failed to show relationship to dose. The body weight of Control offspring was higher than, and body weights at 50, 150 or 500/300 mg/kg/day were lower than the HCD range (males 6.4-6.6 g; females 6.1-6.4 g; 7 studies November 15-May 2019)). Subsequent body weight gain to Day 21 of age was marginally lower than Control at all doses, but the differences only attained statistical significance for females at 300 mg/kg/day (94%). It is noted that the mean body weight of the Control exceeded the HCD range (45.7-49.6 g; 7 studies November 15-May 2019).
Overall body weight gain (Days 1-71) in males receiving 300 mg/kg/day was slightly low, when compared with Control, (88%). Overall body weight gain was slightly high in females receiving 150 mg/kg/day (114% of Control), but was similar to Control at 300 mg/kg/day.
Overall body weight gain of males and females receiving 50 or 150 mg/kg/day was considered to be unaffected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was considered to be unaffected by treatment at 50, 150 or 300 mg/kg/day.
There were some minor variations in food consumption during the 10 week treatment period at 150 or 300 mg/kg/day; however, these were minor and considered not to be toxicologically significant.

Haematological findings:

no effects observed

Description (incidence and severity):

It is considered that the hematological examination in males and females receiving 50, 150 or 300 mg/kg/day at Week 13 of age, revealed no toxicologically significant differences from Control. All differences were confined to one sex, were minor, or lacked dose relationship and were therefore attributed to normal biological variation.
Hemoglobin concentration was marginally high in males receiving 300 mg/kg/day, mean cell hemoglobin concentration was marginally high in males and females receiving 300 mg/kg/day and mean cell hemoglobin was high in females at 300 mg/kg/day (all approximately 103%). Counts of neutrophils and lymphocytes were high in males only at 50, 150 or 300 mg/kg/day (neutrophils 168%, 150% or 138% and lymphocytes 111% or 151% and 132% of Control, respectively) and contributed to the high white cell count in males at these doses (124%, 150% or 133% of Control, respectively); however, none showed dose relationship.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

When compared with Control, the biochemical examination of the blood plasma at Week 13 of age at 50, 150 or 300 mg/kg/day revealed no toxicologically significant differences from Control. All differences were minor, confined to one sex and lacked dose relationship and were therefore attributed to normal biological variation.
In males only and when compared with Control, cholesterol concentration was low at 300 mg/kg/day (83%) and triglyceride concentration at 50, 150 or 300 mg/kg/day were low, without dose relationship (58%, 74% or 63%, respectively) and in females only, alkaline phosphatase activity and urea concentration were high (122% and 116%, respectively) at 300 mg/kg/day.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

When compared with Control, the composition of the urine at Week 13 of age revealed high total protein in males and females receiving 300 mg/kg/day (146% and 206% of Control, respectively).
All other differences from Control were minor, lacked dose-relationship, or confined to one sex and were therefore attributed to normal biological variation. Such differences included the high total potassium concentration in males and females receiving 300 mg/kg/day (both 145%) that was higher in males receiving 150 mg/kg/day (173%) and in females only, total sodium concentration was high at 300 mg/kg/day (209%) and total chloride concentration was high at 50 or 300 mg/kg/day (209% or 204%), but not at 150 mg/kg/day. Urinary volume was higher than Control in males receiving 150 or 300 mg/kg/day and in females receiving 50 or 300 mg/kg/day (134% or 138% and 157% or 167%, respectively); however there was no clear relationship to dose and was thus considered due to chance.

Sexual maturation:

no effects observed

Description (incidence and severity):

Sexual maturation was considered to be unaffected by treatment at 50, 150 or 300 mg/kg/day.
The body weight of males at balano preputial separation at 300 mg/kg/day was marginally low, when compared with Control. Vaginal opening was marginally delayed at 300 mg/kg/day (106%) and the difference in age was only marginally above the degree of accuracy of the assessment (animals were assessed once per day) and there was no dose response; therefore, the delay was considered not to be toxicologically significant.
The period between vaginal opening and first estrus and estrous cycles at Week 11 of age were unaffected at 50, 150 or 300 mg/kg/day.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Ano-genital distance in male and female offspring was unaffected by parental treatment at 50, 150 or 500/300 mg/kg/day.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Male offspring did not develop nipples.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 13 weeks of age and when compared with Control, body weight relative kidney weight was slightly high in males treated at 50, 150 or 300 mg/kg/day (105%, 109% or 122%, respectively), liver weight was marginally high at 300 mg/kg/day (112%) and thyroid weight was marginally high, without relationship to dose at 50, 150 or 300 mg/kg/day (115%, 113% or 113%. Mean body weight relative brain weight showed marginal non dose related variation at 50, 150 or 300 mg/kg/day (107%, 102% or 107% of Control, respectively). In females at 300 mg/kg/day, absolute and body weight relative thyroid weight was slightly high (116%), without relationship to dose and pituitary weight was slightly high (116%).

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination performed on F1 animals after treatment from weaning to 13 weeks of age revealed no test item-related lesions.
The nature, incidence and distribution of all findings were considered incidental and unrelated to test item.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with Dichlorotoluene Mixture were seen in the liver and kidneys of Cohort 1A males and the kidneys of Cohort 1B males
Liver, Centrilobular hypertrophy was seen in males given 150 or 300 mg/kg/day
Summary of treatment related findings in the liver for F1A males
Group/sex 1M 2M 3M 4M
Dose (mg/kg/day) 0 50 150 300
Hypertrophy, Centrilobular
Minimal 0 0 4 9
Slight 0 0 0 8
Total 0 0 4 17
Number of tissues examined 20 20 18ef 19d
d One male (No. 468) was killed for welfare reasons.
e One male (No. 445) was found dead. f One male (No. 452) killed for welfare reasons.
Kidneys
Increased incidence and severity of hyaline droplet accumulation accompanied by basophilic tubules and/or granular casts was seen in males given 50, 150 or 300 mg/kg/day.
Summary of treatment related findings in the kidneys for F1A males
Group/sex 1M 2M 3M 4M
Dose (mg/kg/day) 0 50 150 300
Accumulation, Hyaline Droplets
Minimal 5 12 9 0
Slight 0 0 9 2
Moderate 0 0 0 16
Marked 0 0 0 1
Total 5 12 18 19
Basophilia, Tubular
Minimal 1 2 10 7
Slight 0 0 1 9
Moderate 0 0 0 2
Total 1 2 11 18
Cast(s), Granular
Minimal 0 0 8 8
Slight 0 0 0 7
moderate 0 0 0 2
Total 0 0 8 17
Number of tissues examined 20 20 18ef 19d
d One male (No. 468) was killed for welfare reasons.
e One male (No. 445) was found dead. f One male (No. 452) killed for welfare reasons.

Other effects:

no effects observed

Description (incidence and severity):

All other microscopic findings were considered to be incidental and unrelated to the test item.
Seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted. The qualitative examination of the ovary revealed no abnormality.
The numbers of ovarian follicles and corpora lutea were unaffected by treatment at 50, 150 or 300 mg/kg/day.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

other: Mortality at high dose not due to systemic toxicity

Remarks:

Based on irritancy of test item

Target system / organ toxicity (F1)

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study a No Observed Adverse Effect Level (NOAEL) for systemic toxicity in F0 and F1 male rats, when treated with Dichlorotoluene Mixture was 50 mg/kg/day, due to hyaline droplets with associated degenerative changes (basophilic tubules and granular casts). This alpha 2u globulin associated nephropathy was considered adverse at 150 or 300 mg/kg/day. However, it is acknowledged that this finding is specific to the male rat and is generally considered to be of no relevance to man. Excluding this finding the NOAEL for male rats is 300 mg/kg/day.
The NOAEL for systemic and reproductive toxicology in adult female rats was concluded to be 300 mg/kg/day. The dose of 300 mg/kg/day is also concluded to be the NOAEL for the offspring based on the low observed body weight gain following weaning.
Special consideration should be made when administering Dichlorotoluene Mixture (classified for skin irritancy) by the oral gavage route, due to the low incidence of respiratory distress and death observed at 150 or 300 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the pre- and post-natal effects of Dichlorotoluene Mixture, an industrial chemical, when administered orally, by gavage, to Han Wistar rats. The evaluation included assessment of the integrity and performance of the adult male and female reproductive tract, and systemic toxicity in pregnant and lactating females and in young and adult offspring. The study was requested by the ECHADecision number TPE‑D‑2114394001-60-01/F.

In the F0 generation, three groups of 25 male and 25 female Han Wistar rats receivedDichlorotoluene Mixture at doses of 50, 150 or 500/300 mg/kg bw/day (treatment to Group 4 (500 mg/kg bw/day) was lowered to 300 mg/kg bw/day from Week 11 (commencement of pairing)) at a volume dose of 5 mL/kg/day. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. In the F1 generation, 20 males and 20 females were treated at 50, 150 or 300 mg/kg bw/day from weaning to their scheduled termination, at the same volume-dose as the F0 generation. A similarly constituted Control group received the vehicle, polyethylene glycol 400 (specific gravity 1.125),at the same volume dosethroughout the same period.

F0: Data were recorded onclinical condition, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance. Clinical pathology (hematology, blood chemistry and thyroid‑related hormones), sperm assessment, organ weight, macroscopic pathology and microscopic pathology investigations were performed. 

The F1 generation comprised of two cohorts. Each cohort comprised of 20 male and 20 female progeny from each dose group and were treated withDichlorotoluene Mixture, by oral gavage administration, at doses of 0 (Control), 50, 150 or 300 mg/kg bw/dayfrom weaning (Day 21 of age) until scheduled sacrifice. Cohort A was terminated at approximately Week 13 of age and Cohort B was terminated at approximately Week 14 of age. 

F1: Offspring, data were recorded on clinical condition, litter size and survival, sex ratio, body weight, anogenital distance, nipple count (male offspring on Day 13 of age), organ weights and macropathology (on Day 4 or 22 of age), were assessed. Blood samples were also collected from selected offspring on Day 4 and 22 of age for investigation of thyroid-related hormones. 

F1: Cohort A,data were recorded on clinical condition, body weight, food consumption,sexual maturation, vaginal opening and estrous cycles. Clinical pathology (hematology, blood chemistry, urinalysis and thyroid-related hormones), sperm assessment, ovarian follicle counts,organ weight, macroscopic pathology, microscopic pathologyandimmunophenotypinginvestigations were performed.

F1: Cohort B, data was recorded on clinical condition, body weight, food consumption,sexual maturation, selected organ weights and a targeted set of macroscopic pathology investigations were performed. 

Results

F0 adults and F1 offspring up to day 25 of age

Treatment at 500 mg/kg/day was not tolerated by a small number of animals. Four animals died or were killed for welfare reasons within the ten week pre‑pairing treatment period following brief periods of respiratory distress and two had blocked nasal turbinates. Treatment at 500 mg/kg/day was reduced to 300 mg/kg/day from Week 11; Day 1 (day of pairing). One female receiving 150 mg/kg/day was killed for welfare reasons following signs of respiratory distress.

There were no systemic clinical signs considered to be related to treatment and no effect on body weight gainduring the 18 week treatment period in males and 17 week treatment period in females, including gestation and lactation to Day 21 post-partum. Food consumption was unaffected by treatment.

Estrous cycles, pre‑coital interval, mating performance, fertility, or gestation length and gestation index; the mean number of implantations and litter size, the ratio of males to females or survival indices were unaffected by treatment.

Minor findings in the hematology or blood plasma parameters had no microscopic correlate.

Following parental treatment at 50, 150 or 500/300 mg/kg/day, the clinical condition of the F1 pups remained good. Mean body weights of treated male and female offspring on Day 1 of age were marginally low, without relationship to dose. Subsequent body weight gain was marginally lower than Control and, at formal commencement of the F1 generation, mean body weights were low at 50, 150 or 300 mg/kg/day. Body weight gain improved in males and females at 50 or 150 mg/kg/day and females at 300 mg/kg/day; however body weight gain in males at 300 mg/kg/day remained marginally, low and was considered adverse.

In the F1, anogenital distance was unaffected by parental treatment at 50, 150 or 500/300 mg/kg/day and males did not develop nipples. Serum T4 and TSH concentrations in the adult and offspring on Days 4 and 22 of age were unaffected by treatment.

There was no effect of treatment on the number or motility or morphology of the sperm.

There were no macroscopic findings attributed to treatment.

Selected F1 offspring - Cohorts 1A and 1B

The clinical condition of the F1 animals remained generally good.

Six animals receiving 300 mg/kg/day and two receiving 150 mg/kg/day died, or were killed for welfare reasons, after showing signs of respiratory distress and one animal receiving 150 mg/kg/day was mis-dosed.

Sexual maturation of the F1 and the period between vaginal opening and first estrus, and estrous cycles at Week 11 of age (before termination) were unaffected by treatment at 50, 150 or 300 mg/kg/day.

Serum T4 and TSH concentrations in the adult and Immunophenotyping parameters, measured in spleen leukocytes (both Cohort 1A), were unaffected by treatment at 50, 150 or 300 mg/kg/day.

Pathology of the F0 and F1 in males only

Microscopic examination revealed that oral gavage administration ofDichlorotoluene Mixtureresulted in test item-related changes in the liver and kidneys ofF0 and F1 males. F0 and F1 females were unaffected. In the liver, incidences of centrilobular hypertrophy were dose-related in males treated at 150 or 500/300 or 300 mg/kg/day and correlated with increased liver weights in F0 males. This possibly resulted from disturbance of hepatic metabolic activity and was therefore considered an adaptive response. In the kidney, there was anaccumulation of hyaline droplets in the cytoplasm of the cortical tubular epithelium, accompanied by basophilic tubules, and/or granular casts at 150 or 500/300 mg/kg/day. This showed dose-relationship and were present in the kidneys of F0 and F1 (Cohort A and B) males given 150 or 500/300 or 300 mg/kg/day, and F0 and F1 (Cohort A) males given 50 mg/kg/day. These changes correlated with increased kidney weights seen in F0 males and males given 300 mg/kg/day of the F1 (Cohort A). Hyaline droplets are a common finding in male kidneys, composed of alpha-2u-globulin and in more severe cases, can be associated with degenerative changes (basophilic tubules and granular casts), as seen in this study. Hyaline droplet-mediated nephropathy is considered rat specific and therefore not relevant to humans as little or no alpha-2u-globulin is present in humans.

Conclusion

Based on the results of this study a No Observed Adverse Effect Level (NOAEL) for systemic toxicity in F0 and F1 male rats, when treated withDichlorotoluene Mixturewas 50 mg/kg/day, due to hyaline droplets with associated degenerative changes (basophilic tubules and granular casts). This alpha‑2u‑globulin associated nephropathy was considered adverse at 150 or 300 mg/kg/day. However, it is acknowledged that this finding is specific to the male rat and is generally considered to be of no relevance to man. Excluding this finding the NOAEL for male rats is 300 mg/kg/day.

The NOAEL for systemic and reproductive toxicology in adult female rats was concluded to be 300 mg/kg/day. The dose of 300 mg/kg/day is also concluded to be the NOAEL for the offspring based on the low observed body weight gain following weaning.

Special consideration should be made when administering Dichlorotoluene Mixture (classified for skin irritancy) by the oral gavage route, due to the low incidence of respiratory distress and death observed at 150 or 300 mg/kg/day.