Tall oil, maleated

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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 number: HD0379UD11

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats: Crl: WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.

Number of animals: 88 (40 males and 48 females) + 3 spare males.
A total of 40 females were selected at randomization before initiation of the pretest phase and then based on estrous cyclicity. Any female without at least two regular estrous cycles was replaced by one of the 8 additional females having at least two regular estrous cycles. The supernumerary females were then removed from the study. Estrous cycle data are retained in the raw data but are not reported.

The virgin males were approximately 11 weeks old at the start of treatment (body weight range 324 to 432 g).
The virgin females were approximately 13 weeks old at the start of treatment (body weight range 203 to 244 g).

Justification: one of the rodent species acceptable to the regulatory agencies. Historical control data for the strain are available at the Test Facility.

Animal husbandry
Housing: One air-conditioned room in a barrier protected unit (building K1).
Temperature: 22 + 3 °C (target range).
Relative humidity: >35% (target).
Air changes: At least 10 air changes per hour.
Lighting cycle: 12 hours light (artificial)/12 hours dark (except when required for technical acts).
Environmental conditions were within the targets throughout the study.
Caging: The animals were caged as follows:
Phase Number of animals per cage
Males Females
Pre-mating 5 5
Mating 1 + 1 (housed together)
Gestation 5 1
Lactation - 1 + litter

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).
Diet: 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.
Water: 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.
Bedding: 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.
Enrichment: 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.
Contaminants: No known contaminants were present in the bedding, diet or water at levels which might have interfered with achieving the objective of the study.
Certificates of analysis for the bedding, diet, water and enrichment are maintained in the archives of the Test Facility.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400

Details on exposure:

Method of administration: Gavage using a plastic cannula (Vygon ref 270.08). The cannula was rinsed with water between each administration.Formulations were maintained under continuous stirring for at least 15 minutes before and throughout the dosing procedure.
Volume of administration: 5 mL/kg/day.
Individual dose volumes were calculated using the latest body weight.
Rationale for choice of route of administration: the oral route was selected as this is a potential route of human exposure during manufacture, handling or use of the test substance as specified in the applicable guidelines.

Details on mating procedure:

After 2 weeks of treatment, animals were paired on the basis of one male and one female from the same group for a maximum of 14 days.
The day of mating was confirmed by the presence of sperm in a vaginal smear or a vaginal plug and was recorded and taken as day 0 of gestation (G 0). Mated females were separated from the males once mating had been confirmed and smearing ceased or when the appearance of the female suggests pregnancy from an undetected mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

One set of samples (2 x 0.5g 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:

Frequency: Once daily.

Details on study schedule:

Study initiation date (study plan signed by Study Director): 24 May 2017.
Experimental starting date (randomisation of the animals for the DRF phase): 24 May 2017.
First day of treatment (Day 1): 18 July 2017.
Experimental completion date (last necropsy): 23 September 2017.
Study completion date: date of signature of the final report by the Study Director.

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

Parental animals: Observations and examinations:

Morbidity/mortality: All animals were observed twice daily at the beginning and at the end of each working day (including weekends and public holidays) to detect any which were dead or moribund.

Clinical signs
The animals were observed daily for clinical signs.
To detect any clinical signs or reactions to treatment, the animals were observed before and once after dosing. A full clinical examination was performed on each weighing day. Towards the end of the gestation, females were examined daily for signs of parturition.

Body weight
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 consumption of males was recorded weekly during the pre-mating period.
Food consumption of females was recorded for the following periods:
- weekly during the pre-mating period
- gestation: Days 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 20
- lactation: Days 1 to 4, 4 to 7 and 7 to 13.

Functional Tests
Animals examined: 5 selected animals/sex/group.
Frequency: Males: on Day 35 (last day of dosing), the day before scheduled necropsy.
Females: on Day 12 (penultimate day of dosing)* or Day 13 of lactation (last day of dosing), 2 or 1 day(s) before scheduled necropsy, respectively.

*for logistical reasons, the locomotor activity in an open field for group 3 female nos. 154 and 160 were monitored on Lactation Day 12.

Tests performed: Auditory reflex.
Pupillary reflex.
Righting reflex.
Fore-limb grip strength.
Locomotor activity in an open field test. Activity was monitored by a video image analysis system (Videotrack supplied by Viewpoint, Lyon, France). The arena is divided into nine equal invisible regions; the time spent by the animal in each type of region (i.e. corner, centre or lateral) was recorded. Motor activity was divided into three categories: ambulatory activity (in which the centre of the image moves at more than 7 cm/sec), small movements (including grooming etc.) and inactivity. The proportion of time spent engaged in each type of activity and the total distance travelled by the rat were calculated.

Oestrous cyclicity (parental animals):

Daily vaginal smears were performed to determine the stage of oestrus beginning 14 days prior to treatment, the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal smears continued for any female with no evidence of copulation until termination of the mating period.
On the day of scheduled necropsy, a vaginal smear was taken to determine the stage of the estrous cycle and allow correlation with histopathology of female reproductive organs.

Litter observations:

Offspring were examined once daily (including weekends and public holidays) to detect any which were dead or moribund.
The following was recorded for each F1 litter:
- number of pups born (live and dead);
- external abnormalities of the pups;
- number, weight and sex of pups alive on PND 1, 4, 7 and 13.
- Anogenital distance (normalized to the cube root of body weight) on PND 1
- presence of areola/nipples on PND 13 for all males in each litter.

The size of each litter was adjusted to 8 pups on PND 4 by eliminating extra pups by random selection to yield 4 male and 4 female pups per litter where possible.
Blood samples were collected from two surplus pups where possible, pooled and used for determination of serum T4 levels, see section 6.13.1).
No pups were eliminated when litter size dropped below the culling target (8 pups/litter). When there was only one pup available above the culling target, only one pup was eliminated and used for blood collection for possible serum T4 assessments.

Postmortem examinations (parental animals):

All adult males and females were weighed before necropsy (except any found dead).
Animals were killed by carbon dioxide inhalation followed by exsanguination
All animals (including any found dead and females showing signs of parturition difficulties or total litter death) were submitted to necropsy procedures including an examination of following:
- external surface
- all orifices
- cranial cavity
- thoracic and abdominal cavities and organs and their contents
- the carcass.

Any abnormalities observed were recorded and preserved in an appropriate fixative.
For females sacrificed before parturition (including any found dead after the first day of pairing), the ovaries and uterus were removed and examined including examination of the placentae. The following data were recorded:
- pregnancy status
- number of corpora lutea
- number of intrauterine implantations
-number of live embryos
-number of intrauterine deaths (resorption sites).
The uterus of all adult females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites. The number of corpora lutea was counted.
Any foetuses were examined externally where possible and discarded.

Postmortem examinations (offspring):

On PND 4 and PND 13, pups were killed by intraperitoneal injection of sodium pentobarbitone (CEVA Santé Animale).
All pups (including any found dead or killed moribund) were necropsied. Any external abnormalities observed were recorded but not preserved. For any pups found dead or killed moribund, the stomach was examined for the presence of milk and defects or cause of death was evaluated, if possible.
Each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs.

Statistics:

Statistical analysis was performed, where appropriate, by the data acquisition software, as follows:
The best transformation for the data (none, log or rank) was determined depending upon:
- the kurtosis of the data
-the probability of the Bartlett's test for homogeneity of the variances and
-an assessment of whether the size of the groups are approximately equal or not.
Non- or log-transformed data were analyzed by parametric methods.
Rank transformed data were analyzed using non-parametric methods.
Data were then analyzed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analyzed by parametric or non-parametric Dunnett's test to look for significant differences from the control group. Selected incidence data were analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.The locomotor activity in an open field, fore-limb grip strength, estrous cycle and pre-coital interval data were analysed using a SAS softwarepackage. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.

Reproductive indices:

Pre-coital interval (in days): sum of days until successful insemination / number of inseminated females

Male and female copulation index (in %): number of inseminated females / number of paired animals x 100

Male and female fertility index (in %): number of pregnant females / number of inseminated females x 100


Pre-birth loss (in %): number of implantations - number of offspring born / number of implantations x 100

Offspring viability indices:

Live birth index (in %): number of pups born alive / number of pups born x 100


Viability index (in %): number of pups alive on PND 4 / number of pups alive at birth x 100


Lactation index (in %): number of pups alive on PND 13 / number of pups alive on PND 4 (after culling) x 100


Sex ratio (proportion of male pups in %): number of males / number of pups x 100

Clinical signs:

effects observed, non-treatment-related

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.
During gestation, a slightly lower mean body weight gain was noted for females given 100 and 400 mg/kg/day (-8.2 and -4.5% respectively). This was considered incidental as it occurred without any dose-relationship.
Despite a slightly lower body weight gain between L0 and L4 in the 200 and 400 mg/kg/day groups (-13.8 and -27.4%, respectively), there was an overall dose-related higher mean body weight gain during the lactation period in the treated groups (+9, +18 and +30% in the 100, 200 and 400 groups, respectively between L1 and L13) when compared with the control group.
Terminal mean body weight of treated females were therefore comparable with, or slightly higher than, that of the control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Consistent with the higher body weight gain, there was slightly higher mean food consumption in all treated groups for males during the dosing period and for females during the premating and gestation periods when compared with the control group.
There was slightly lower mean food consumption during the lactation periods in all treated female groups mainly over the first days after parturition when compared with the control group. This finding was considered incidental as it occurred without any dose relationship.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

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:

effects observed, non-treatment-related

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:

not examined

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, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related histopathological changes were observed in the liver, as summarised in the following text table.
Text Table. Liver - Incidence of Treatment-Related Histopathologic Findings
Males Females
Dosage group 0 100 200 400 0 100 200 400
Number of animals/group 5 0 0 5 5 0 0 5
Centrilobular vacuolation 0 - - 5 0 - - 1
Minimal - - - 4 - - - 1
Slight - - - 1 - - - -
Glycogen-like deposits 1 - - 5 0 - - 2
Minimal 1 - - 5 - - - 2

a = Number of tissues examined from each group.

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.
There was no microscopic evidence of hepatocellular degeneration associated with the vacuolation, which was therefore considered non-adverse. This was further suggested by the absence of any treatment-related change in liver clinic-pathological parameters.
There were no treatment-related changes in all the other organs.
Additionally, no pathological were observed in the organs examined microscopically in non-pregnant females (nos. 131, 151, 153, 158 and 176) and males that failed to sire (nos. 104, 121, 141, 143, 148 and 166). Male no. 166 treated at 400 mg/kg/day was mated with female no. 176 and failed to sire. At microscopic evaluation, there was minimal bilateral tubular degeneration/atrophy in the testes and minimal cell debris in the epididymides and a relationship to the failure to sire could not be completely ruled out. Degeneration/atrophy of the seminiferous tubules in this male was considered unrelated to treatment in view of its isolated occurrence at 400 mg/kg/day and because it can be observed in untreated rats, as described by Creasy et al. (2012).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

T4 Hormone analysis
No differences in Total T4 levels were noted among the different groups of F0 males or among the different groups of PND 13 pups.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment with the test item in any group.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no test item-related effect on mating performance in any group; all paired animals mated. All mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle) with the exception of one (no. 155) given 200 mg/kg/day with a pre-coital interval of 5 days. The mean pre-coital interval was therefore normal (less than 4 days) in all groups.
There was no test item-related effect on fertility. Most mated females became pregnant with the exception of 1 female in each of the 100 and 400 mg/kg/day groups (female nos. 131 and 176, respectively) and 3 females in the 200 mg/kg/day group (female nos. 151, 153 and 158). The lower number of pregnant females in the mid dose group was considered incidental in the absence of a similar finding in the high dose group.

Key result

Dose descriptor:

NOAEL

Effect level:

> 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive performance

Clinical signs:

no effects observed

Description (incidence and severity):

There were no pup observations that suggested any association with maternal treatment.
Incidental clinical signs were noted such as haematoma, hairloss, cyanosis, cold to touch, thinness or weakness.
There was no test item-related effect on sex ratio in any group. The mean percentage of males per litter was slightly lower in the 400 mg/kg/day group (39.7%) when compared with the control group (50.7%) but remained within the historical control data range (39.4 to 56.9%). This was therefore considered incidental.
There were no pup observations that suggested any association with maternal treatment.
Incidental clinical signs were noted such as haematoma, hairloss, cyanosis, cold to touch, thinness or weakness.
There was no test item-related effect on sex ratio in any group. The mean percentage of males per litter was slightly lower in the 400 mg/kg/day group (39.7%) when compared with the control group (50.7%) but remained within the historical control data range (39.4 to 56.9%). This was therefore considered incidental.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item related effect on mean live litter size or pup viability in any group.
There were 2 pups found dead between birth and Day 4 before culling in the 400 mg/kg/day group compared with none in the control and lower dose groups. However, the viability index in the high dose group (97.7%) remained within the historical control data range (94.4 to 100 %) so there was no toxicological significance. Thereafter, there was no pup found dead or missing in any group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was a non-dose-related slightly lower mean pup body weight at birth in all treated groups (6.45, 6.24 and 6.39 g in the 100, 200 and 400 mg/kg/day, respectively) compared with the control (7.05 g). However, the mean values remained within the historical control data range with the exception of the male pups in the 200 mg/kg/day group (6.31 g compared with historical control data range of 6.38 to 7.38 g). The mean value in this group was disproportionately lowered by one female (no.160) with a markedly low mean pup body weights (4.5g for males and 4.3g for females). This finding in the intermediate dose group was therefore considered incidental.

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

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Other effects:

no effects observed

Description (incidence and severity):

No differences in Total T4 levels were noted among the different groups of PND 13 pups.
Anogenital distance (normalized for body weight) in male and female pups was not affected by treatment.
There were no test item-related effects on areola/nipple retention. None of the male pups examined had nipples observed on PND 13.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no reproductive effects observed at highest dose level

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

400 mg/kg bw/day (nominal)

Treatment related:

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.

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 analyzed once during the study to assess accuracy and homogeneity.

The concentrations analyzed 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.

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.

 

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)

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

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil, maleated

EC Number:

268-859-6

EC Name:

Tall oil, maleated

Cas Number:

68152-93-2

Molecular formula:

UVCB

IUPAC Name:

3,7-dimethyl-14,16-dioxo-19-(propan-2-yl)-15-oxapentacyclo[10.5.2.0²,¹¹.0³,⁸.0¹³,¹⁷]nonadeca-13(17),18-diene-7-carboxylic acid; 8-(7-hexyl-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)octanoic acid

Test material form:

liquid

Details on test material:

Lot No.: HD0258QH13

Specific details on test material used for the study:

Batch no. : HD0379UD11

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose group

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

intermediate dose group

Doses / concentrations 3

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

Examinations

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)

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

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.