Reaction mass of m-terphenyl and o-terphenyl

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

other: combined study (repeated dose and reproduction/developmental toxicity screening)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-10 to 2017-11-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-10 to 2017-11-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2017Reach
- Expiration date of the lot/batch: February 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a refrigerator (2 to 8 °C). Stored under Nitrogen.
- Solubility and stability of the test substance in the solvent/vehicle: Formulations were assessed daily by Envigo pharmacy.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item and corn oil (vehicle) was heated to 75°C in a water bath. For each formulation, the required amount of test item was mixed with approximately 70% of the final volume of vehicle and magnetically stirred whilst heating to approximately 75% in a water bath until uniformly mixed. The remaining volume of vehicle was added and the formulation magnetically mixed in the water bath. The formulation was cooled gradually to room temperature, continually stirring.

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

Species:

rat

Strain:

other: Crl:CD(SD) rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Males 10 to 11 weeks old. Females 12 to 13 weeks old.
- Weight at study initiation: Males 336 to 403 g. Females 234 to 296 g.
- Fasting period before study: no data
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Pre-pairing: up to five animals of one sex per cage; Pairing: one male and one female; Males after mating: up to five animals; Gestation: one female; Lactation: one female + litter
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet. Non-restricted (removed overnight before blood sampling for hematology and blood chemistry investigations).
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Non-restricted.
- Acclimation period: Males: 7 days before commencement of treatment. Females: 7 days before commencement of estrous cycle evaluation.

DETAILS OF FOOD AND WATER QUALITY: The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24ºC.
- Humidity (%): Monitored and maintained within the range of 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The test item and corn oil (vehicle) was heated to 75°C in a water bath. For each formulation, the required amount of test item was mixed with approximately 70% of the final volume of vehicle and magnetically stirred whilst heating to approximately 75% in a water bath until uniformly mixed. The remaining volume of vehicle was added and the formulation magnetically mixed in the water bath. The formulation was cooled gradually to room temperature, continually stirring. Formulations were prepared in ascending group order. Formulations were assessed daily by Envigo pharmacy, and if crystalline, reheated to produce a liquid formulation. No crystallisation was noted.

- VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment and on Day 12 of lactation were analysed via HPLC for achieved concentration of the test item.
The mean concentrations were within applied limits +10/-15%, confirming the accuracy of formulation. The percentage difference from mean values remained within ±3%, confirming precision of the analysis with the exception in Week 1, Group 2 where the percentage difference from mean was ±7.17%.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and a 2-week mating period in both sexes plus 1 week post-mating in males, or the entire gestation period as well as 14 days of lactation in females.

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: no satellite group
- Post-exposure recovery period in satellite groups: not applicable

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
Cages were inspected twice 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.

BODY WEIGHT: Yes
- Time schedule for examinations:
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 before pairing. Days 0, 7, 14 and 20 after mating. Day 1, 4, 7 and 13 of lactation. On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food supplied to each cage of F0 animals, that remaining and an estimate of any spilled was recorded as follows: Weekly, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording: Days 0-7, 7-14 and 14-20 after mating, Days 1-4, 4-7 and 7-13 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food at termination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: five males and five females per group
- Parameters given below were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food at termination.
- Animals fasted: Yes
- How many animals: five males and five females per group
- Parameters given below were examined.

URINALYSIS: No data.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: during Week 5 of treatment; female: at Day 7-9 of lactation
- Dose groups that were examined: each group
- Battery of functions tested: sensory activity / grip strength / motor activity. The following measurements, reflexes and responses were recorded: Approach response, Pinna reflex, Auditory startle reflex, Tail pinch response, Grip strength, Motor activity.

OTHER: Thyroid hormone analysis, Estrous Cycle, reproduction indices, clinical observation of litters/pups and their necropsy findings

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. 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.

HISTOPATHOLOGY: Yes (see table)

Other examinations:

Thyroid hormone analysis:
- Thyroid stimulating hormone (TSH)
- Thyroxine (T4)

Statistics:

Cochran-Armitage test, Chi-squared test, Dunnett’s test, Fisher’s Exact test, Linear-by-linear test, Shirley’s test, Williams’ test (details see below)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In a few male and female animals given 300 mg/kg/day, chin rubbing and/or salivation were noted on isolated occasions post dose, and on one occasion prior to dosing. There were no other signs associated with dosing at this or lower doses.
The distribution of minor signs noted at the detailed physical examination and arena observations showed no test item related effects.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were two decedent animals, both in females given 300 mg/kg/day.
Female 68: The cause of death was undetermined but in the absence of similar findings in other females given 300 mg/kg/day is considered of uncertain relationship to the test item.
Female 67: This mortality is considered an accidental death and not test item related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weight gain over Weeks 0-5 for males given 100 or 300 mg/kg/day was lower, but not statistically significant, than the concurrent control group (0.84x and 0.78x Control respectively).
There were no test item related effects on body weight over Weeks 0-5 in the males given 30 mg/kg/day.
There were no test item related effects on body weight over Weeks 0-2, prior to pairing, or during gestation or lactation in females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were no test item related effects on food consumption of males.
In females given 300 mg/kg/day, group mean food consumption during lactation was slightly yet statistically significantly lower than the controls (0.81x Control). Food consumption of females from all groups was similar during the first 2 weeks of treatment and during gestation and there were no effects on food consumption during lactation in females given 30 or 100 mg/kg/day.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males and females given 300 mg/kg/day group mean values for haemoglobin (0.93x and 0.91x Control respectively), red blood cells (0.92x and 0.92x Control respectively) and, for males only, haematocrit (0.92x Control) were statistically significantly lower than the concurrent control group.
In males given 300 mg/kg/day, group mean values for white blood cells (1.75x Control), mainly due to an increase in lymphocytes (1.91x Control), were statistically significantly higher than the concurrent control group. In females given 300 mg/kg/day group mean values for lymphocytes was also statistically significantly higher (1.75x Control) than the controls.
In males given 300 mg/kg/day, group mean values for red cell distribution width (1.17x Control) and activated partial thromboplastin time (1.33x Control) were statistically significantly higher than the concurrent control group. There were no test item related effects in animals given 30 or 100 mg/kg/day.
Group mean serum concentration of thyroxine (T4) at termination in adult males given 300 mg/kg/day were lower than the concurrent control (0.59x Control).
There were no effects on serum concentration of thyroxine (T4) in adult males given 30 or 100 mg/kg/day, adult females from all treated groups, or offspring on Day 13 of age.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males and females given 100 and 300 mg/kg/day, group mean aspartate aminotransferase levels were statistically significantly lower than the concurrent control group (0.72x and 0.38x Control for males and 0.48x and 0.48x Control for females, respectively). Low aspartate aminotransferase is not a biologically relevant measure and as such this is considered to be incidental.
In males given 300 mg/kg/day, group mean cholesterol level was statistically significantly higher than the concurrent control group (1.61x Control).
There were no further test item related effects on blood chemistry.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There were no test item related effects on the sensory reactivity observations and grip strength.
In females given 100 and 300 mg/kg/day, group mean high and low beam scores were higher than the concurrent control group, with dose-relationship evident (high beam 1.45x and 1.91x Control; low beam 1.40x and 1.48x Control respectively). Statistical significance was attained at 300 mg/kg/day for several of the 6-minute intervals and the total high beam scores and at the 48-minute interval high beam score. However most of the high beam scores, including the total score, for females were within the historical control data range, and for one of the 6 minute intervals, the control group was below the historical data range, and although some of the low beam scores were above the historical data range only one of the intervals attained statistical significance. As such this change is considered of uncertain relationship to the test item.
There were considered to be no test item related effects on the group mean activity scores for males.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weight adjusted kidney weights in males given 100 and 300 mg/kg/day were slightly but statistically significantly higher than the concurrent controls (1.14x and 1.17x Control respectively).
Group mean body weight adjusted (absolute for females given 300 mg/kg/day) liver weights in males given 100 mg/kg/day and males and females given 300 mg/kg/day were slightly but statistically significantly higher than the concurrent controls (1.24x and 1.48x Control for males and 1.37x Control for females, respectively). There were no other effects on organ weights.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic examination performed after scheduled termination showed no test item related lesions.
The incidence and distribution of all findings were considered to be unrelated to treatment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment were found in the liver, thyroid, and kidneys.
Hepatocellular hypertrophy was seen in all males and three females given 300 mg/kg/day and in one male and three females given 100 mg/kg/day.
Diffuse follicular cell hypertrophy was seen in four males and females given 300 mg/kg/day and in three males and two females given 100 mg/kg/day.
Minimal or slight cortical basophilia was seen in all males treated with 300 mg/kg/day compared with only one control male.
All other histological changes were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous Cycles, Pre-Coital Interval, Mating Performance, Fertility and Gestation Length, and Estrous Stage at Termination:
All females assigned to the study had regular estrous cycles prior to the start of treatment.
There were no test item related findings on estrous cycles in the first 2 weeks of treatment prior to pairing.
There were no test item related effects on mating performance or fertility.
The pre-coital interval was 1-4 days for the majority of parings, indicating mating occurred at the first estrous opportunity.
All pairings showed evidence of mating, apart from one male/female pairing at 100 mg/kg/day and of these, conception occurred in all but 1 female given 100 mg/kg/day. A total of 9, 10, 8 and 10 females were pregnant in the control group and in females given 30, 100 or 300 mg/kg/day, respectively.
There was a slight shift in gestation length in females given 300 mg/kg/day with 2 females with a 22 day gestation length compared with 4 control females, and 7 females with a 23 day gestation length compared with 5 control females. In the absence of any further effects on mating performance this is considered unlikely to be test item related.
The gestation index was 100% in all groups with the exception of females given 300 mg/kg/day where one female died on Gestation Day 23 prior to commencement of parturition.
Prior to termination all females smeared were in diestrus. There were no test item related effects.

Incidential findings:
In the kidneys, mineralisation was seen in the cortical tubules of three control females, three females given 30 mg/kg/day and four females given 100 mg/kg/day, with none evident in the females given 300 mg/kg/day, or in males. This mineralisation was also seen in the myocardium and cardiac vasculature, and in the glandular portion of the stomach. In some of these animals, microscopic mineralisation could be correlated macroscopically to multifocal dark areas within the glandular stomach and pale areas on the heart.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general, systemic

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects around parturition

Critical effects observed:

not specified

Conclusions:

The no-observed-adverse-effect level (NOAEL) of o,m-terphenyl reaction mass for systemic toxicity and also for reproductive/developmental toxicity was considered to be 100 mg/kg/day, based on effects around parturition at 300 mg/kg/day.

Executive summary:

This study was performed according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) and GLP. The purpose of this study was to assess the general systemic toxic potential of o,m-terphenyl reaction mass in rats, including a screen for reproductive/developmental effects and assessment of endocrine related endpoints, when administered to Sprague Dawley rats by oral gavage administration for at least 4 weeks.

In the liver, hepatocellular hypertrophy was recorded in males and females and this microscopic finding correlated with increased mean liver weights in both sexes at 100 and 300 mg/kg/day. Hepatocellular hypertrophy accompanied by increases in liver weight is usually a result of hepatic enzymatic induction.

Follicular cell hypertrophy in the thyroid was considered as a secondary effect of test item induced hepatic enzymatic induction of the liver, which may have been responsible for the lower thyroxine (T4) levels seen in males given 300 mg/kg/day. The findings in the liver and thyroid are therefore considered adaptive (Richardson 2010, Zabka et al 2011).

In the kidneys, a minimal or slight cortical basophilia was seen at a higher incidence in males given 300 mg/kg/day and this correlated with increased kidney weights in males at this dose. No changes were observed in the clinical pathology parameters indicating impaired renal function in treated animals. The distribution of mineralisation in the kidneys of control and treated group females indicates alteration in calcium/phosphorus homeostasis. In this study the reproductive performance of the control group (cyclical activity, mating performance, fertility rate, litter size, offspring survival, growth and clinical condition) was within the expected background range. The presence of mineralisation in the kidneys, heart and stomach of some animals from control and treated groups is considered very unlikely to have compromised evaluation or interpretation. The treatment-related effect (tubular basophilia) in treated female kidneys was also seen in males, who were completely unaffected by the mineralisation complex, substantiating the accuracy of interpretation of any test item related effects. All of these outcomes support the power of the study to detect test item related effects.

This study included a screen for reproductive/developmental effects. Estrous cycles, precoital interval, mating performance, fertility and gestation length were unaffected by treatment. The mean number of implantations, litter size and live birth index at 300 mg/kg/day were slightly lower than controls and the historical control range. At 300 mg/kg/day, one female was sacrificed on Gestation Day 23 due to apparent dystocia, a second female exhibited total litter loss by Lactation Day 1 and a third female had a high pup loss from birth to Lactation Day 1. As such it is considered there is a slight effect on reproductive/development around parturition. No test-item related microscopic pathological changes were observed in the male or female reproductive organs. The clinical condition of the offspring, their post birth growth and survival, sex ratio, ano-genital distance on Day 1 of age and male nipple counts on Day 13 of age showed no adverse effects of parental treatment. There were also no signs in the decedent offspring, or offspring at termination on Day 13 of age that were considered to be related to parental treatment.

The study design also included an assessment of endocrine relevant endpoints. This included the measurement of the hormone thyroxine (T4) in adult males and females and in offspring at Day 13 of age, by evaluating changes in adult organ weight and gross organ pathology of endocrine-sensitive organs and, because some developmental stages (e.g. gestational and neonatal) are particularly sensitive to endocrine effects, an external examination of all offspring, measurement of the ano-genital distance of offspring on Day 1 of age and nipple counts for male offspring on Day 13 of age. Lower thyroxine levels in males given 300 mg/kg/day was likely a consequence of/associated with the thyroid follicular cell hypertrophy, but as no lowering of thyroxine levels was noted in females given 300 mg/kg/day the effect on males is of uncertain toxicological significance. No effect of treatment was evident on the circulating levels of thyroxine in offspring on Day 13 of age. No significant changes were identified at the microscopic examination of adrenal and pituitary glands and the reproductive organs. All offspring were macroscopically normal; in particular no effects were seen on the external genitalia. Ano-genital distance was slightly longer in male offspring from parents given 300 mg/kg/day but this direction of change is not indicative of any feminising effects. No male offspring had any nipples. It was therefore concluded that, in the context of this study, o,m-terphenyl reaction mass showed no evidence of affecting endocrine relevant endpoints.

The changes in aspartate aminotransferase in this study were considered of no toxicological significance as the direction of response was lower than controls, and this marker is raised when indicating tissue damage.

The no-observed-adverse-effect level (NOAEL) of o,m-terphenyl reaction mass for systemic toxicity and also for reproductive/developmental toxicity was considered to be 100 mg/kg/day, based on effects around parturition at 300 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of m-terphenyl and o-terphenyl

EC Number:

904-797-4

Molecular formula:

C36H28

IUPAC Name:

Reaction mass of m-terphenyl and o-terphenyl

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2017Reach
- Expiration date of the lot/batch: February 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a refrigerator (2 to 8°C). Stored under Nitrogen.
- Solubility and stability of the test substance in the solvent/vehicle: Formulations were assessed daily by Envigo pharmacy.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item and corn oil (vehicle) was heated to 75°C in a water bath. For each formulation, the required amount of test item was mixed with approximately 70% of the final volume of vehicle and magnetically stirred whilst heating to approximately 75% in a water bath until uniformly mixed. The remaining volume of vehicle was added and the formulation magnetically mixed in the water bath. The formulation was cooled gradually to room temperature, continually stirring.

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

Test animals

Species:

rat

Strain:

other: Crl:CD(SD) rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Males 10 to 11 weeks old. Females 12 to 13 weeks old.
- Weight at study initiation: Males 336 to 403 g. Females 234 to 296 g.
- Fasting period before study: no data
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Pre-pairing: up to five animals of one sex per cage; Pairing: one male and one female; Males after mating: up to five animals; Gestation: one female; Lactation: one female + litter
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet. Non-restricted (removed overnight before blood sampling for hematology and blood chemistry investigations).
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Non-restricted.
- Acclimation period: Males: 7 days before commencement of treatment. Females: 7 days before commencement of estrous cycle evaluation.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24ºC.
- Humidity (%): Monitored and maintained within the range of 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test item and corn oil (vehicle) was heated to 75°C in a water bath. For each formulation, the required amount of test item was mixed with approximately 70% of the final volume of vehicle and magnetically stirred whilst heating to approximately 75% in a water bath until uniformly mixed. The remaining volume of vehicle was added and the formulation magnetically mixed in the water bath. The formulation was cooled gradually to room temperature, continually stirring. Formulations were prepared in ascending group order. Formulations were assessed daily by Envigo pharmacy, and if crystalline, reheated to produce a liquid formulation. No crystallisation was noted.

- VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg body weight

Details on mating procedure:

- M/F ratio per cage: 1:1 from within the same treatment groups.
- Length of cohabitation: Up to two weeks.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment and on Day 12 of lactation were analysed via HPLC for achieved concentration of the test item.
The mean concentrations were within applied limits +10/-15%, confirming the accuracy of formulation. The percentage difference from mean values remained within ±3%, confirming precision of the analysis with the exception in Week 1, Group 2 where the percentage difference from mean was ±7.17%.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and a 2-week mating period in both sexes plus 1 week post-mating in males, or the entire gestation period as well as 14 days of lactation in females.

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment (if not random): Before the commencement of treatment, study allocation was revised to ensure all females had regular estrus cycles and to reduce inter/intra group body weight variation by replacement with spare animals of suitable weight.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cages were inspected twice 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.

BODY WEIGHT: Yes
- Time schedule for examinations: 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 before pairing. Days 0, 7, 14 and 20 after mating. Day 1, 4, 7 and 13 of lactation. On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied to each cage of F0 animals, that remaining and an estimate of any spilled was recorded as follows: Weekly, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording: Days 0-7, 7-14 and 14-20 after mating, Days 1-4, 4-7 and 7-13 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
-Dry smears: For 15 days before pairing using cotton swabs.
Wet smears: Using pipette lavage during the following phases:
- For 14 days before treatment; animals that failed to exhibit 4-5 cycles were not allocated to the study.
- After pairing until mating.
- Female showing no evidence of mating – following completion of the pairing period females were separated from the male and vaginal smearing was continued for up to five days.
- For four days before scheduled termination (Days 11 to 14 of lactation).

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations:
For the assessment of the testes, 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.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

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), presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after Week 5 investigations completed.
- Maternal animals:
F0 females failing to mate: Day 25 after last day of pairing.
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females whose litter died before Day 13: On or after day the last offspring died.
F0 females: Day 14 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Adult animals were sacrificed by carbon dioxide asphyxiation with subsequent exsanguination. 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Examined externally, if found to be normal offspring were discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues were retained in an appropriate fixative.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

Cochran-Armitage test, Chi-squared test, Dunnett’s test, Fisher’s Exact test, Linear-by-linear test, Shirley’s test, Williams’ test

Reproductive indices:

Percentage mating (%) = Number of animals mating / Animals paired * 100
Conception rate (%) = Number of animals achieving pregnancy / Animals mated * 100
Fertility index (%) = Number of animals achieving pregnancy / Animals pairing * 100
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 number of live litters born / number pregnant * 100

Offspring viability indices:

Post-implantation survival index (%) = Total number of offspring born / Total number of uterine implantation sites * 100
Live birth index (%) = Number of live offspring on Day 1 after littering / Total number of offspring born * 100
Viability index (%) = Number of live offspring on Day 4 / Number live offspring on Day 1 after littering * 100
Lactation index (%) = Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after selection for thyroid hormone bleed) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In a few male and female animals given 300 mg/kg/day, chin rubbing and/or salivation were noted on isolated occasions post dose, and on one occasion prior to dosing. There were no other signs associated with dosing at this or lower doses.
The distribution of minor signs noted at the detailed physical examination and arena observations showed no test item related effects.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were two decedent animals, both in females given 300 mg/kg/day.
Female 68: The cause of death was undetermined but in the absence of similar findings in other females given 300 mg/kg/day is considered of uncertain relationship to the test item.
Female 67: This mortality is considered an accidental death and not test item related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weight gain over Weeks 0-5 for males given 100 or 300 mg/kg/day was lower, but not statistically significant, than the concurrent control group (0.84x and 0.78x Control respectively). There were no test item related effects on body weight over Weeks 0-5 in the males given 30 mg/kg/day. There were no test item related effects on body weight over Weeks 0-2, prior to pairing, or during gestation or lactation in females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were no test item related effects on food consumption of males.
In females given 300 mg/kg/day, group mean food consumption during lactation was slightly yet statistically significantly lower than the controls (0.81x Control). Food consumption of females from all groups was similar during the first 2 weeks of treatment and during gestation and there were no effects on food consumption during lactation in females given 30 or 100 mg/kg/day.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males and females given 300 mg/kg/day group mean values for haemoglobin (0.93x and 0.91x Control respectively), red blood cells (0.92x and 0.92x Control respectively) and, for males only, haematocrit (0.92x Control) were statistically significantly lower than the concurrent control group. In males given 300 mg/kg/day, group mean values for white blood cells (1.75x Control), mainly due to an increase in lymphocytes (1.91x Control), were statistically significantly higher than the concurrent control group. In females given 300 mg/kg/day group mean values for lymphocytes was also statistically significantly higher (1.75x Control) than the controls. In males given 300 mg/kg/day, group mean values for red cell distribution width (1.17x Control) and activated partial thromboplastin time (1.33x Control) were statistically significantly higher than the concurrent control group.
There were no test item related effects in animals given 30 or 100 mg/kg/day.
Group mean serum concentration of thyroxine (T4) at termination in adult males given 300 mg/kg/day were lower than the concurrent control (0.59x Control).
There were no effects on serum concentration of thyroxine (T4) in adult males given 30 or 100 mg/kg/day, adult females from all treated groups, or offspring on Day 13 of age.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males and females given 100 and 300 mg/kg/day, group mean aspartate aminotransferase levels were statistically significantly lower than the concurrent control group (0.72x and 0.38x Control for males and 0.48x and 0.48x Control for females, respectively). Low aspartate aminotransferase is not a biologically relevant measure and as such this is considered to be incidental. In males given 300 mg/kg/day, group mean cholesterol level was statistically significantly higher than the concurrent control group (1.61x Control).
There were no further test item related effects on blood chemistry.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There were no test item related effects on the sensory reactivity observations and grip strength.
In females given 100 and 300 mg/kg/day, group mean high and low beam scores were higher than the concurrent control group, with dose-relationship evident (high beam 1.45x and 1.91x Control; low beam 1.40x and 1.48x Control respectively). Statistical significance was attained at 300 mg/kg/day for several of the 6-minute intervals and the total high beam scores and at the 48-minute interval high beam score. However most of the high beam scores, including the total score, for females were within the historical control data range, and for one of the 6 minute intervals, the control group was below the historical data range, and although some of the low beam scores were above the historical data range only one of the intervals attained statistical significance. As such this change is considered of uncertain relationship to the test item.
There were considered to be no test item related effects on the group mean activity scores for males.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment were found in the liver, thyroid, and kidneys.
Hepatocellular hypertrophy was seen in all males and three females given 300 mg/kg/day and in one male and three females given 100 mg/kg/day.
Diffuse follicular cell hypertrophy was seen in four males and females given 300 mg/kg/day and in three males and two females given 100 mg/kg/day.
Minimal or slight cortical basophilia was seen in all males treated with 300 mg/kg/day compared with only one control male.
All other histological changes were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females assigned to the study had regular estrous cycles prior to the start of treatment. There were no test item related findings on estrous cycles in the first 2 weeks of treatment prior to pairing.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related effects on mating performance or fertility.
The pre-coital interval was 1-4 days for the majority of parings, indicating mating occurred at the first estrous opportunity.
All pairings showed evidence of mating, apart from one male/female pairing at 100 mg/kg/day and of these, conception occurred in all but 1 female given 100 mg/kg/day. A total of 9, 10, 8 and 10 females were pregnant in the control group and in females given 30, 100 or 300 mg/kg/day, respectively.
There was a slight shift in gestation length in females given 300 mg/kg/day with 2 females with a 22 day gestation length compared with 4 control females, and 7 females with a 23 day gestation length compared with 5 control females. In the absence of any further effects on mating performance this is considered unlikely to be test item related.
The gestation index was 100% in all groups with the exception of females given 300 mg/kg/day where one female died on Gestation Day 23 prior to commencement of parturition.
Prior to termination all females smeared were in diestrus. There were no test item related effects.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general, systemic

2

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects around parturition

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item related effects related to parental treatment in the few clinical signs noted in the offspring from birth to Day 13 of age.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related effects related to parental treatment on body weight gain to Day 13 of age.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item related effects related to parental treatment noted at macroscopic examination of the offspring.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Litter Size, Sex Ratio and Survival Indices (see also tables 1 and 2 below):
In females given 300 mg/kg/day there was a statistically significantly lower mean number of implantations (0.84x Control), which was associated with a statistically significantly lower total litter size at birth (0.81x Control). Group mean live litter size on Day 1 was lower than the control (0.75x Control), although this was influenced by one of the litters where 5/13 pups died between birth and Day 1. Excluding this litter from the group means live litter size on Day 1 was 0.78x Control, a similar magnitude of response to the number of implantations and total litter size. Body weight gain from birth was similar to the controls. There was also, at 300 mg/kg/day, a lower live birth index (not statistically significant) and one case of total litter loss (Day 1 of lactation). There was no test item related effect on sex ratio or survival post birth.
There were no test item related effects related to parental treatment at 30 or 100 mg/kg/day on the number of implantations, litter size or sex ratio. The offspring survival indices were similar in all groups with no adverse effects.

Ano-genital distance:
In male offspring from parental treatment at 300 mg/kg/day there was a slight but statistically significantly longer ano-genital distance, adjusted for body weight (1.07x Control). All group mean ano-genital distances, including controls, were outside the historical data range. However as the magnitude of the change was small (0.3 mm) and was not indicative of any feminising effect (shorter ano-genital distance) this is considered to be of no toxicological significance.
There were no test item related effects related to parental treatment at 30 or 100 mg/kg/day, or in female offspring at 300 mg/kg/day, on the ano-genital distance on Day 1 of age, corrected for the body weight.

Offspring nipple count:
There were no nipples/areolae noted in any male offspring on Day 13.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Based on:

test mat.

Sex:

male/female

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Litter size - group mean values (F1)

Dose (mg/kg/day)		Implantations	Total @	Live on Day
				Before cull		After cull
				1	4	4	7	13
Statistics test		Wi	Wi	Wi	Wi
0	Mean	15.8	14.8	14.7	14.7	12.9	12.9	12.9
	SD	2.11	0.97	1.00	1.00	1.05	1.05	1.05
	N	9	9	9	9	9	9	9
30	Mean	14.9	14.6	14.5	14.5	12.6	12.5	12.5
	SD	1.37	1.51	1.51	1.51	1.51	1.35	1.35
	N	10	10	10	10	10	10	10
100	Mean	15.1	14.1	14.0	13.9	12.0	11.9	11.8
	SD	1.25	2.17	2.00	1.89	1.69	1.64	1.67
	N	8	8	8	8	8	8	8
300	Mean	13.3*	12.0**	11.0**	11.0**	10.0	10.0	10.0
	SD	2.63	2.31	2.45	2.45	1.53	1.53	1.53
	N	7	7	7	7	7	7	7

@ - Includes offspring that died prior to the designated Day 1 of age

** p<0.01

Table 2: Offspring survival indices - group mean values (F1)

Dose (mg/kg/day)		Post implantation survival index (%)		Live birth index (%)		Viability index (%)	Lactation index (%)
						Day 4	Day 13
Statistics test		Wi		Ch		Ch
0	Mean	90.7		99.3		100.0	100.0
	SD	6.45		2.22		0.00	0.00
	N	9		9		9	9
	N<100%	7		1		0	0
30	Mean	97.3		99.3		100.0	99.3
	SD	3.45		2.11		0.00	2.11
	N	10		10		10	10
	N<100%	4		1		0	1
100	Mean	92.3		99.3		99.2	98.1
	SD	7.78		2.08		2.21	5.44
	N	8		8		8	8
	N<100%	5		1		1	1
300	Mean	90.7		92.4		100.0	100.0
	SD	7.95		14.05		0.00	0.00
	N	7		7		7	7
	N<100%	5		3		0	0

Applicant's summary and conclusion

Conclusions:

The no-observed-adverse-effect level (NOAEL) of o,m-terphenyl reaction mass for systemic toxicity and also for reproductive/developmental toxicity was considered to be 100 mg/kg/day, based on effects around parturition at 300 mg/kg/day.

Executive summary:

This study was performed according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) and GLP.

The purpose of this study was to assess the general systemic toxic potential of o,m-terphenyl reaction mass in rats, including a screen for reproductive/developmental effects and assessment of endocrine related endpoints, when administered to Sprague Dawley rats by oral gavage administration for at least 4 weeks.

In the liver, hepatocellular hypertrophy was recorded in males and females and this microscopic finding correlated with increased mean liver weights in both sexes at 100 and 300 mg/kg/day. Hepatocellular hypertrophy accompanied by increases in liver weight is usually a result of hepatic enzymatic induction.

Follicular cell hypertrophy in the thyroid was considered as a secondary effect of test item induced hepatic enzymatic induction of the liver, which may have been responsible for the lower thyroxine (T4) levels seen in males given 300 mg/kg/day. The findings in the liver and thyroid are therefore considered adaptive. (Richardson 2010, Zabka et al 2011).

In the kidneys, a minimal or slight cortical basophilia was seen at a higher incidence in males given 300 mg/kg/day and this correlated with increased kidney weights in males at this dose. No changes were observed in the clinical pathology parameters indicating impaired renal function in treated animals. The distribution of mineralisation in the kidneys of control and treated group females indicates alteration in calcium/phosphorus homeostasis. In this study the reproductive performance of the control group (cyclical activity, mating performance, fertility rate, litter size, offspring survival, growth and clinical condition) was within the expected background range. The presence of mineralisation in the kidneys, heart and stomach of some animals from control and treated groups is considered very unlikely to have compromised evaluation or interpretation. The treatment-related effect (tubular basophilia) in treated female kidneys was also seen in males, who were completely unaffected by the mineralisation complex, substantiating the accuracy of interpretation of any test item related effects. All of these outcomes support the power of the study to detect test item related effects.

This study included a screen for reproductive/developmental effects. Estrous cycles, precoital interval, mating performance, fertility and gestation length were unaffected by treatment. The mean number of implantations, litter size and live birth index at 300 mg/kg/day were slightly lower than controls and the historical control range. At 300 mg/kg/day, one female was sacrificed on Gestation Day 23 due to apparent dystocia, a second female exhibited total litter loss by Lactation Day 1 and a third female had a high pup loss from birth to Lactation Day 1. As such it is considered there is a slight effect on reproductive/development around parturition. No test-item related microscopic pathological changes were observed in the male or female reproductive organs. The clinical condition of the offspring, their post birth growth and survival, sex ratio, ano-genital distance on Day 1 of age and male nipple counts on Day 13 of age showed no adverse effects of parental treatment. There were also no signs in the decedent offspring, or offspring at termination on Day 13 of age that were considered to be related to parental treatment.

The study design also included an assessment of endocrine relevant endpoints. This included the measurement of the hormone thyroxine (T4) in adult males and females and in offspring at Day 13 of age, by evaluating changes in adult organ weight and gross organ pathology of endocrine-sensitive organs and, because some developmental stages (e.g. gestational and neonatal) are particularly sensitive to endocrine effects, an external examination of all offspring, measurement of the ano-genital distance of offspring on Day 1 of age and nipple counts for male offspring on Day 13 of age. Lower thyroxine levels in males given 300 mg/kg/day was likely a consequence of/associated with the thyroid follicular cell hypertrophy, but as no lowering of thyroxine levels was noted in females given 300 mg/kg/day the effect on males is of uncertain toxicological significance. No effect of treatment was evident on the circulating levels of thyroxine in offspring on Day 13 of age. No significant changes were identified at the microscopic examination of adrenal and pituitary glands and the reproductive organs. All offspring were macroscopically normal; in particular no effects were seen on the external genitalia. Ano-genital distance was slightly longer in male offspring from parents given 300 mg/kg/day but this direction of change is not indicative of any feminising effects. No male offspring had any nipples. It was therefore concluded that, in the context of this study, o,m-terphenyl reaction mass showed no evidence of affecting endocrine relevant endpoints.

The changes in aspartate aminotransferase in this study were considered of no toxicological significance as the direction of response was lower than controls, and this marker is raised when indicating tissue damage.

The no-observed-adverse-effect level (NOAEL) of o,m-terphenyl reaction mass for systemic toxicity and also for reproductive/developmental toxicity was considered to be 100 mg/kg/day, based on effects around parturition at 300 mg/kg/day.