Tall oil, maleated

Endpoint summary

Description of key information

Under the experimental conditions of an OECD 422 study with 35 days exposure to males and >50 days exposure to females, the daily oral (gavage) administration of the test item, EnvaMul 600, to the rat during pre-mating (males and females), during pregnancy and lactation (females) at doses of 100, 200 and 400 mg/kg/day induced no parental, reproduction or developmental toxicity. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 400 mg/kg was derived.

In addition a subchronic oral toxicity study with rats was performed, according to OECD TG 408 protocol with doses of 0, 100, 300 and 1000 Mg/kg bw/d, respectively. The systemic NOAEL was set conservatively to 300 mg/kg bw/d as the centrilobular hepatocellular hypertrophy in combination with the multifocal hepatocellular necrosis in a single male at 1000 mg/kg/day was considered adverse. However, for local effect a NOAEL of 100 mg/kg bw/d based on irritating effects to the gastrointestinanl tract at 300 and 1000 mg/kg bw/d was set.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

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)

Version / remarks:

The study was performed in accordance with the original study plan and subsequent amendments with the following deviations:
- Thyroid and parathyroid glands were lost at necropsy for group 2 male no. 129.
- On Day -16 (pretest) during mortality check, control female no. 115 was found in the cage of female nos. 116 to 120. Consequently, these animals were housed in group of 6 for a few hours.

These deviations were considered not to have affected the outcome or the achievement of the study objectives.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Batch no. : HD0379UD11

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: the virgin males were approximately 11 weeks old at the start of treatment - The virgin females were approximately 13 weeks old at the start of treatment
- Weight at study initiation: males: body weight range 324 to 432 g; females: body weight range 203 to 244 g
- Fasting period before study:
- Housing: Group housed males and females and individual housed females, including females during mating and with litters, were housed in plastic cages in compliance with European Regulations (Directive 2010/63/EU). Cellulose bedding (Serlab, Montataire, France) or dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood, analysed at least twice a year for chemical and bacterial contaminants.
A small amount (handful) of shredded paper was provided as enrichment for all animals. Any isolated animals had free access to a wooden gnaw block (Aspen Bricks, Le comptoir des sciures, France).
Furthermore, tissue paper was also provided as enrichment for females towards the end of gestation.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Males: 8 days between arrival and the start of treatment, Females: 8 days between arrival and start of pre-test estrous cycle smears.

DETAILS OF FOOD AND WATER QUALITY: Rat pelleted commercial complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
Adult animals were fasted overnight before sampling for clinical laboratory determinations.
Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via an automatic watering system or bottles). Water is analysed twice a year for bacterial and chemical contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3 °C
- Humidity (%): >35%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark (except

Route of administration:

oral: gavage

Details on route of administration:

Gavage using a plastic cannula (Vygon ref 270.08). The cannula was rinsed with water between each administration.

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400 Supplier: Merck Batch number: K47000103605

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
Dose levels were selected based on the results of the DRF phase.
Administration of EnvaMul 600 at doses of 300 and 500 mg/kg/day in the female Han Wistar rat for 10 days was associated with a slight reduced body weight gain at 500 mg/kg/day and hypersalivation at both doses of 300 and 500 mg/kg/day. Due to the decrease in body weight gain in 2 out of 3 animals at 500 mg/kg/day between days 5 and 10 compared to the first few days of dosing, and following discussion with the Sponsor, the dose of 500 mg/kg/day was considered to be too high for a longer dosing period, including a gestation phase.
Doses of 100, 200 and 400 mg/kg/day were selected for the subsequent reproduction/developmental toxicity screening test study in the rat.

- VEHICLE
- Concentration in vehicle: The test item was prepared as a suspension in the vehicle at concentrations of 20, 40 and 80 mg/mL according to Standard Operating Procedures of the Test Facility.
- Amount of vehicle (if gavage): 5 mL/kg/day (volume of administration)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

One set of samples (2 x 0.5 g per sampling position) for accuracy and homogeneity evaluation was dispatched at room temperature protected from light to the Test Site for analysis.
The second set of formulation samples kept at the Test Facility was not analysed and was discarded following the issue of the final study report.
Reference for analysis: Analysis of the samples (Charles River Laboratories Den Bosch BV. project 519107) was performed according to a method validated at the Test Site (Charles River Laboratories Den Bosch BV. project 519106).
The accuracy of preparation was considered acceptable if the mean measured concentrations are 85-115 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤10 %.

Duration of treatment / exposure:

Duration: Males were dosed for 35 days, i.e. 14 days prior to mating, throughout the mating period and up to the day prior to necropsy. The first day of dosing is designated as Day 1.
Females that delivered were treated for at least 51 days, i.e. during 14 days prior to mating (the first day of dosing is designated as Day 1), the variable time to conception (the first day of gestation is designated as G 0), the duration of the pregnancy and 13 days after delivery (the first day of birth is designated as L 0), up to and including the day before scheduled necropsy. Females which failed to deliver were treated for at least 40 days.

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose group

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

intermediate dose group

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

high dose group

No. of animals per sex per dose:

1. Control: 10 males and 10 females
2. Low dose: 10 males and 10 females
3. Intermediate dose: 10 males and 10 females
4. High dose: 10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected based on the results of the DRF phase (see addendum 1). Administration of EnvaMul 600 at doses of 300 and 500 mg/kg/day in the female Han Wistar rat for 10 days was associated with a slight reduced body weight gain at 500 mg/kg/day and hypersalivation at both doses of 300 and 500 mg/kg/day. Due to the decrease in body weight gain in 2 out of 3 animals at 500 mg/kg/day between days 5 and 10 compared to the first few days of dosing, and following discussion with the Sponsor, the dose of 500 mg/kg/day was considered to be too high for a longer dosing period, including a gestation phase.
Doses of 100, 200 and 400 mg/kg/day were selected for the subsequent reproduction/developmental toxicity screening test study in the rat.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Male and females were weighed during pre-test, on the first day of exposure (prior to the first exposure) and weekly thereafter during pre-mating and mating periods.
Mated females were weighed:
- on Days 0, 6, 9, 12, 15, 18 and 20 of gestation
- on Days 1, 4, 7 and 13 of lactation.


FOOD EFFICIENCY: No


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the pre-mating period (Day 14)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: first 5 animals/sex/group
- Parameters checked in table:
Haemoglobin
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
Packed cell volume
Red blood cell count
Mean corpuscular volume
Reticulocyte count
Platelet count
Total white blood cell count
Differential white blood cell count.
Prothrombin time
Activated partial thromboplastin time
Fibrinogen.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the pre-mating period (Day 14)
- Animals fasted: Yes
- How many animals: first 5 animals/sex/group
- Parameters checked in table: Sodium
Potassium
Chloride
Calcium
Inorganic phosphorus
Glucose
Urea
Total cholesterol
Total bilirubin
Total protein
Albumin
Globuline (calculated)
Albumin/globulin ratio (calculated)
Creatinine
Alkaline phosphatase
Aspartate aminotransferase
Alanine aminotransferase
Bile acids.
were examined.

URINALYSIS: no

NEUROBEHAVIOURAL EXAMINATION: Yes
- Dose groups that were examined: first 5 animals/sex/group
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength, motor activity (open field)

IMMUNOLOGY: no

OTHER: T4 hormone analysis

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)

Clinical signs:

no effects observed

Description (incidence and severity):

Hypersalivation associated or not with abnormal foraging and/or pedalling was noted for all males and majority of females in the control group from Day 17 and in treated groups from Day 10, mainly immediately after dosing. These signs, also noted at a comparable incidence between treated and control groups were considered to be a physiological response rather than a sign of systemic toxicity; considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to the palatability of the vehicle and/or the test-item.
Test-item related red coloured urine associated or not with pallor, was noted for up to 5 females up to 5 days between lactation days 2 and 6 at 400 mg/kg/day. These transient findings were considered as not adverse.
Isolated incidental clinical signs were noted such as bent tail, piloerection, abnormal vocalisation, sunken eye, soft feces, thinness and broken tooth. These changes were considered incidental or associated with parturition.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control female (no. 114) was found dead on Gestation Day 21 (G 21) after dosing. Dark lungs, liquid in the trachea and soiled nose were noted at necropsy. This death was therefore attributed to a dosing error.
There was no other unscheduled death in any group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males: There was a non-dose-related higher mean body weight gain during the dosing period in all treated groups when compared with the control.
Females: Mean body weight gain in all treated groups was higher compared with the control during the premating period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no differences noted in haematological and coagulation parameters between control and treated rats of either sex that were considered to be toxicologically relevant.
Although some differences in mean values attained statistical significance compared with the concurrent control, mainly for males, such as decreases in mean red blood cell count, polymorphonuclear neutrophils and monocytes, they were considered of no toxicological significance since there was no dose-related trend and/or in view of the low magnitude. In addition, mean values were within the historical control range.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no differences noted in serum clinical chemistry parameters between control and treated rats of either sex that were considered to be toxicologically relevant.
Dose related higher mean triglyceride concentration for both sexes in treated groups compared with the concurrent control was likely to be related to the nature of the test item (mixture of fatty acids) and was therefore not considered to be toxicologically relevant since values remains within the historical control data range.
Although some differences in mean values attained statistical significance for males and/or females compared with the concurrent control, such as decreases in protein, albumin and aspartate aminotransferase concentrations, they were considered of no toxicological significance in view of the low magnitude. In addition, mean values were within the historical control range.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Test-item related red coloured urine associated or not with pallor, was noted for up to 5 females up to 5 days between lactation days 2 and 6 at 400 mg/kg/day. These transient findings were considered as not adverse.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.
There were no test item-related effects on motor activity (open field) for either sex in any group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment-related organ weight changes were observed in the liver.
In both sexes, the group mean absolute and relative liver weight in treated groups were generally higher than in the control group, with dose-related trend.
In rats treated at 400 mg/kg/day, the higher liver weight was considered to correlate microscopically with hepatocellular vacuolation and increased glycogen deposits. These histopathological changes were generally of minimal severity.
The group mean absolute and relative weights of the uterus in females treated at 400 mg/kg/day were approximately 20% lower than in the control group. At the standard examination of the transversal section of each uterine horn, no correlating histopathological changes were observed. The numbers of implantation traces in control females and those treated at 400 mg/kg/day were comparable and no clear correlation with the uterus weight was observed on an individual animal basis. Therefore, any correlation between the treatment and the slightly lower uterus weight in females treated at 400 mg/kg/day was uncertain.
All the other organ weight differences between control and treated groups were considered incidental and/or secondary to the slightly increased final body weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment-related histopathological changes were observed in the liver.
The treatment-related liver changes were generally at a low severity, and both the incidence and severity were slight higher in males. The hepatocellular vacuolation was characterized by the presence of a large cytoplasmic, round to oval vacuole, which was either empty or exhibited a weakly stained eosinophilic content in males. In female no. 178, bright eosinophilic material/droplets were observed in the cytoplasmic vacuoles.
The vacuolation was accompanied by minimal glycogen-like hepatocellular deposits, which was sporadically observed in control rats.
These changes were considered to correlate with the slight increase in liver weight in male and females rats treated at 400 mg/kg/day compared with control and the other treated groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Critical effects observed:

no

Conclusions:

Under the experimental conditions of the study, the daily oral (gavage) administration of the test item, EnvaMul 600, to the rat during pre-mating (males and females), during pregnancy and lactation (females) at doses of 100, 200 and 400 mg/kg/day induced no parental, reproduction or developmental toxicity.
Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 400 mg/kg was derived.

Executive summary:

The objectives of the study were to evaluate the potential toxic effects of the test item, EnvaMul 600, when administered to rats for a minimum of 28 days and the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. Parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No-Observed-Adverse-Effect-Levels (NOAELs) were evaluated.

Procedures

The test item, EnvaMul, was administered by oral gavage at dose levels of 100, 200 and 400 mg/kg/day to groups of 10 male and 10 female Wistar rats. A fourth group received the vehicle (polyethylene glycol 400). Males were treated for 35 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females that delivered were treated for at least 51 days, i.e. for 2 weeks prior to mating, during mating, during pregnancy, and during 13 days of lactation.

The following observations and examinations were evaluated: mortality / morbidity, clinical signs, functional observations and locomotor activity, body weight, food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4, macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: copulation and fertility indices, pre-coital time, number of implantation sites, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy). Formulations were analysed once during the study to assess accuracy and homogeneity.

Results

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations and the formulations of Group 2 and Group 4 were homogeneous.

There was no test item-related mortality in any group.

There were no test item-related clinical signs.

Despite a slightly lower body weight gain between L0 and L4 in the 200 and 400 mg/kg/day groups, there was an overall dose-related higher mean body weight gain during the lactation period in the treated groups when compared with the control group. Terminal mean body weights of treated females were therefore comparable with, or slightly higher than, that of the control group.

There were non-adverse test item-related changes on the haematological and serum clinical chemistry parameters.

No differences in Total T4 levels were noted among the different groups of adult males or among the different groups of PND 13 pups.

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex, grip strength or open field tests were observed for the males or females at any dose level.

There were no test item-related effects on mating performance of the males and females or on fertility in any group.

There were 9, 9, 7 and 9 females in the control, 100, 200 and 400 mg/kg/day groups, respectively, that successfully completed delivery with liveborn pups.

There was an incidentally lower number of implantation and consequently litter size in the 400 mg/kg/day group when compared with control.

There were no test item-related effects on pre-birth loss and pup viability.

There was an incidentally lower mean pup body weight in all treated groups compared with the control.

There were no test item-related effects on areola/nipple retention. None of the examined male pups had nipples observed at PND 13.

There was no test item-related effect on anogenital distance (normalized for body weight) for the male and female pups.

At 400 mg/kg/day, treatment-related non-adverse changes were observed in the liver, such as minimal or slight centrilobular hepatocellular vacuolation and increased incidence of minimal glycogen deposits. These changes correlated with the slight increase in liver weight in both sexes.

The microscopic evaluation of the liver in rats treated at 100 or 200 mg/kg/day is suggested in order to evaluate the presence of treatment-related liver changes at these doses.

Conclusion

Under the experimental conditions of the study, the daily oral (gavage) administration of the test item, EnvaMul 600, to the rat during pre-mating (males and females), during pregnancy and lactation (females) at doses of 100, 200 and 400 mg/kg/day induced no parental, reproduction or developmental toxicity. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 400 mg/kg was derived.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In an OECD 422 study with 35 days exposure of males and >51 days exposure of females, no adverse effects were seen at the high dose group (400 mg/kg bw/d); likewise, the NOAEL of 300 mg/kg bw/d for systemic toxicity was found in a subchronic study (OECD TG 408) and thus, the substance does not require classification for specific target organ toxicity, repeat exposure (STOT RE) according to CLP (Regulation EC No. 1272/2008).