Tall oil, maleated

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No adverse effects on fertility were seen in a GLP OECD 422 study. The NOAEL for fertility effects was set to the highest dose applied (400 mg/kg bw/d).

Link to relevant study records

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.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

No developmental effects were seen in a GLP OECD 422 study. The NOAEL for developmental toxicity was set to the highest dose applied (400 mg/kg bw/d). In a developmental toxicity study according to OECD TG 414, dosed at 0, 100, 300 and 1000 mg/kg bw/d, 1000 mg/kg bw/d was found to be the NOAEL for developmental toxicity in the absence of deleopmental effects.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Identification: Maleated TOFA
Batch (Lot) Number: XF09SS
Expiry Date: 12 July 2022
Physical Description: Dark brown viscous liquid
Purity/Composition: 100%

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han rats (Crl: WI(Han). )

Details on test animals or test system and environmental conditions:

Age at Initiation of Dosing: 11 - 15 weeks old
Body Weight Range at Initiation of Dosing: 181 – 254 g
Source (Main + DRF study): Charles River Deutschland, Sulzfeld, Germany
Number of Acclimation days: 5 – 6 days
Environmental Conditions: The actual daily mean temperature during the study period was 19 to 21 °C with an actual daily mean relative humidity of 50 to 76%. The values that were outside the targeted mean humidity range occurred for 5 days with a maximum of 76% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.
Caging: Polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS-J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals will be individually housed. These housing conditions will be maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the animals will be kept will be documented in the study records. Cages will be arranged on the racks according to a Latin-square model.
Cage Identification: Color-coded cage card indicating at least Test Facility Study No., group, animal identification number.
Animal Enrichment: For psychological/environmental enrichment and nesting material, animals will be provided with paper and with aspen wooden sticks, except when interrupted by study procedures/activities. Results of analysis for contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants that would interfere with the objectives of the study.
Environmental Conditions: The target conditions for animal room environment will be as follows:
Temperature: 18 to 24 °C
Humidity: 40 to 70%
Light Cycle: 12-hours light and 12-hours dark (may be interrupted for designated procedures).
Ventilation: At least 10 air changes per hour.
Any variations to these conditions will be evaluated and maintained in the raw data.
Food: Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany Type: Pellets (alternate diet may be provided on individual animal basis as warranted as approved by the Study Director).
Frequency: Ad libitum, except during designated procedures.
Analysis: Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Water:
Type: Municipal tap water.
Frequency/Ration: Freely available to each animal via water bottles.
Analysis: Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
Veterinary Care: Veterinary care will be available throughout the course of the study and animals will be examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, will be documented in the study records.
In the event that animals show signs of illness or distress, the responsible veterinarian may make initial recommendations about treatment of the animal(s) and/or alteration of study procedures, which must be approved by the Study Director. All such actions will be properly documented in the study records and, when appropriate, by Study Plan amendment. Treatment of the animal(s) for minor injuries or ailments may be approved without prior consultation with the Sponsor Representative when such treatment does not impact fulfillment of the study objectives. If the condition of the animal(s) warrants significant therapeutic intervention or alterations in study procedures, the Sponsor Representative will be contacted, when possible, to discuss appropriate action. If the condition of the animal(s) is such that emergency measures must be taken, the Study Director and/or attending veterinarian will attempt to consult with the Sponsor Representative prior to responding to the medical crisis, but the Study Director and/or veterinarian has authority to act immediately at his/her discretion to alleviate suffering. The Sponsor Representative will be fully informed of any such events.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

supplied by (Merck, Darmstadt, Germany), Specific Gravity: 1.036

Details on exposure:

The objectives of this study are to determine the potential of Maleated TOFA to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Days 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity will be evaluated.
A Dose Range Finder (Test Facility Reference No. 20252893) was conducted to select dose levels for the Main study. Four groups of 6 females were exposed to 0, 300, 600 and 1000 mg/kg/day Days 6 to 20 post-coitum inclusive by oral gavage.
Homogeneity and stability of the test item under test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 20252890).
The dose levels were selected based on the results of an Acute Oral Toxicity Study, on the DRF phase of an OECD422 combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (ECHA dossier) and on a 14 day repeated dose study (Test Facility Reference No. 20252891) with Maleated TOFA in Wistar Han rats by oral gavage. In the Acute Oral Toxicity Study, the LD50 value was found to be above 2000 mg/kg in female rats. In the DRF phase of the OECD422 combination study, where 2 groups of 3 female Han Wistar rats were treated at 300 and 500 mg/kg/day for 10 days, animals were noted with a slight reduced body weight gain (2/3 animals) at 500 mg/kg/day and hypersalivation at both
doses of 300 and 500 mg/kg/day. In the OECD422 combination study, a parental, reproduction and developmental NOAEL was derived of at least 400 mg/kg/day.
In the 14 days repeated dose study, 2 groups of 3 female Wistar Han rats were treated by oral gavage for 14 days. Dose levels were 500 and 1000 mg/kg/day. Animals were noted with salivation and a slight increase in liver weight on both dose levels. No further adverse effect were noted. Based on these results, and in consultation with the Sponsor, dose levels for the current Dose Range Finder study were selected at 300, 600 and 1000 mg/kg/day.
Dosing of the DRF was initiated on 14 Apr 2021. The in-life phase of the DRF was completed on 29 Apr 2021. The test item and vehicle were administered to the appropriate animals by once daily oral gavage from Days 6 to 20 post-coitum, inclusive at 0, 300, 600, and 1000 mg/kg bw/d with 6 females per dose group. No mortality occurred during the study period. Based on the results of the DRF, selected dose levels for the Main study were 100, 300 and 1000 mg/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

An ultra performance liquid chromatographic method with mass spectrometric detection (UPLC-MS) for the quantitative analysis of he test item in vehicle was developed and applied (project 20252890).
Organic phase was acetonitrile and aqueous phase consisted of 10 mM ammonium acetate in water. The instrument used was Acquity UPLC system with an Acquity TQD mass spectrometer. The column used was an Acquity UPLC BEH C18 50 mm * 2.1 mm i.d., dp 1.7 μm at 40 °C. Injection volume was 2 μL and flow was 0.6 mL/min; MS detection by ESI-.
Calibration solutios were determined in duplicates for the required analytical range. Accuracy of calibrations and test solution in the range of 85 - 115% were given and repeatability of ≤10% was given (validity criteria).
coefficient r = >0.99 with a deviation ≤15%
Further details on analytical method can be found in the analytical report, appended to the final study report.

Details on mating procedure:

The test system, justification of test system, animal identification and environmental acclimation were identical as for the RF study.
Number of Females: 22 (time-mated) per dose group.
Number of Fetuses Expected: ~ 12 fetuses/female.
Untreated females will be mated at the Supplier and will be at Day 0 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).
At arrival, animals will be assigned to groups at random and animals will be identified using a chip. Animals in poor health will not be assigned to groups.

Duration of treatment / exposure:

The test item and vehicle was administered to the appropriate animals by one daily oral gavage from Day 6 to Day 20 post-coitum, inclusive.
Dose pot identification via Provantis was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Frequency of treatment:

Once daily

Duration of test:

daily exposure by oral gavage from Day 6 to Day 20 post-coitum, inclusive

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

control, vehicle only

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

low dose group

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

mid dose group

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

high dose group, maximum dose recommended

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

The study design followed OECD TG 414 protocol.
The preparation of test item was identical as for the DRFstudy. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 24 hours after preparation of the formulation. For analytical verifications see above.
Homogeneity and stability of the test item under test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 20252890).
Test System
The test system, justification of test system, animal identification and environmental acclimation was identical as for the DRF study. Number of Females in DRF study: 24 (time-mated) and in main study 88 (22 per dose group).
Untreated females were mated at the animal supplier and were at Day 0 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating). At arrival, animals were assigned to groups (22 animals per dose group) at random and animals were identified using a chip. Animals in poor health will not be assigned to groups.
At Day 6 daily exposure by gavage started until Day 20, inclusive.
In-life Procedures, Observations, and Measurements - General In-life Assessments – F0-Animals
Mortality: All DRF animals at least twice daily (morning and afternoon) beginning upon arrival through termination/release / Animals will be observed within their cage unless necessary for identification or confirmation of possible findings.
Cageside Observations: All DRF animals; at least once daily, starting on Day 6 post-coitum onwards up to the day prior to necropsy. Directly post-dose. / Animals will be observed within their cage unless necessary for identification or confirmation of possible findings. Cage debris will be examined to detect premature birth, if applicable.
Detailed Clinical Observations: All DRF animals; on Days 2, 6, 13 and 21 post-coitum / Animals are removed from the cage.
Individual Body Weights: All DRF animals; on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum. / In order to monitor the health status animals may be weighed more often.
Food Consumption: All DRF animals; over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum. / Quantitatively measured.
Terminal Procedures – F1-Generation / F1-animals
Viable fetus of dam surviving until scheduled necropsy on Day 21 post-coitum: External Examination, Body Weight and External Sex Determination
Non-viable fetus of dam surviving until scheduled necropsy on Day 21 post-coitum: External Examination, Body Weight and External Sex Determination
Late resorption: External Examination
Fetus of dam not surviving until scheduled necropsy on Day 21 post-coitum (Dams that are found dead, sacrificed before planned necropsy or that started to deliver): External Examination
Live fetuses will be euthanized by decapitation. In case decapitation might affect a malformation, the fetus will be euthanized by interscapular injection of sodium pentobarbital.
• Malformed fetuses will be collected and fixed in the most appropriate fixative (based on type of malformation).
• Malformed late resorptions will be collected and fixed in 10% buffered formalin.
Fetuses and late resorptions without malformations will be discarded.

Maternal examinations:

Comprising
- mortality
- clinical observations
- body weight & body weight gain
- food consumption
- thyroid hormone analysis
- thyroid weight
- macroscopic evaluations
- microscopic evaluations of thyroid gland
- maternal pregnancy data

Ovaries and uterine content:

see above

Blood sampling:

see above

Fetal examinations:

Comprising
- litter size
- sex ratio
- fetal body weights
- fetal anogenital distance
- fetal morphological examinations
- external malformations & variations
- visceral malformations & variations
- skeletal malformations & variations

Statistics:

The procedures of constructed variables, statistical analysis, computerized systems, amendments and deviations, and retention of records were identical in the Main study with DRF study. All statistical analyses were performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions were summarized and statistically analyzed as indicated below according to sex and occasion.
Parametric/Non-parametric: Levene’s test will be used to assess the homogeneity of group variances. The groups will be compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons will be conducted using Dunnett’s or Dunn’s test, respectively.
Non-Parametric: The groups will be compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons will be conducted using Dunn’s test.
ANCOVA: The data corresponding to a response variable of interest and to a related covariate will be submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons will be conducted using Dunnett’s test.
Incidence:A Fisher’s exact test will be used to conduct pairwise group comparisons of interest.

Indices:

Incidence:A Fisher’s exact test will be used to conduct pairwise group comparisons of interest.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were noted during the observation period.
At 1000 mg/kg/day, two females were noted with abnormal breathing sounds on either Days 7-8 post-coitum or on Day 21 post-coitum. Given the low incidence, this was considered not toxicologically relevant.
Erected fur was noted for one female at 100 and 300 mg/kg/day each and for three females at 1000 mg/kg/day, but also for two control females, on 3-7 days between Days 10-20 post-coitum. In absence of a clear dose response relation, this finding was regarded unrelated to treatment with the test item.
Salivation was observed after dosing among most animals at 1000 mg/kg/day and a few animals at 300 and 100 mg/kg/day on 1-3 days between Days 8-12 post-coitum. A higher incidence was noted with increasing dose. Considering the nature and minor severity of this effect and its time of occurrence (i.e. after dosing) it was regarded to be a physiological response rather than a sign of systemic toxicity. Skin scab findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable in gavage study

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, body weight gain was slightly lower compared to control between Days 9-12 and 18-21 post-coitum (not achieving statistical significance at the later time point). This resulted in an overall mean body weight gain over the treatment period (Days 6-21 post-coitum) that was 14% lower compared to the control mean. At the end of the treatment period on Day 21 post-coitum, mean body weight was 4% lower than control (no statistical significance was achieved).
Noteworthy, Female No. 79 showed slight body weight loss (5 gram) between Days 18-21 post-coitum. This female had a normal litter (i.e. 11 live fetuses, no resorptions).
Mean body weight gain corrected for gravid uterus was decreased at 1000 mg/kg/day (23% lower than control; not statistically significant). This could mainly be attributed to Female Nos. 73, 78 and 79 (adjusted BWG of 0.8, -1.9 and -1.0 gram, respectively). After exclusion of these females, the mean body weight gain corrected for gravid uterus at 1000 mg/kg/day was improved, but still lower than control (11% lower than control).
Noteworthy, a single female of the control group (No. 16) was also noted with a low body weight gain corrected for gravid uterus (adjusted BWG of 2.4 gram).
At 300 and 100 mg/kg/day, mean body weights, body weight gain and weight gain corrected for gravid uterus were considered to be in the same range as control over the treatment period. Mean gravid uterus weights was slightly decreased at 1000 mg/kg/day (11% lower than control; not statistically significant). This decrease in gravid uterus weight was mainly attributable to females with relative few live fetuses (Nos. 75 and 80 with 3 and 4 live fetuses, respectively). Mean gravid uterus weight at 300 and 100 mg/kg/day was considered to be in the same range as control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, mean food consumption was decreased between Days 9-12 post-coitum (9% lower than control) and Days 18-21 post-coitum (7% lower than control; not statistically significant).
At 300 and 100 mg/kg/day, mean food consumption was considered to be in the same range as control.

Food efficiency:

not examined

Description (incidence and severity):

provided ad libitum

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

not examined

Description (incidence and severity):

provided ad libitum

Ophthalmological findings:

not examined

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean serum levels of total triiodothyronine (T3) showed an apparent dose-related trend towards a decrease across the dose groups, but reached statistical significance at 1000 mg/kg/day only (0.8x of control). Mean T3 remained within historical control range across the dose groups. Mean serum levels of total thyroxine (T4) and thyroid stimulating hormone (TSH) were considered to be unaffected by treatment with the test item up to 1000 mg/kg/day.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in thyroid gland weights. There were no test item-related gross observations in the thyroid glands.
All recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations in the thyroid glands.
All recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations in the thyroid glands.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean numbers of corpora lutea, implantation sites, early and late resorptions, and pre- and post-implantation loss in the control and test groups were considered similar and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The mean numbers of corpora lutea, implantation sites, early and late resorptions, and pre- and post-implantation loss in the control and test groups were considered similar and in the range of normal biological variation.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean numbers of corpora lutea, implantation sites, early and late resorptions, and pre- and post-implantation loss in the control and test groups were considered similar and in the range of normal biological variation.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

All females, except for Female No. 46 (300 mg/kg/day), were pregnant and had live fetuses at scheduled necropsy. Therefore, the number of females with viable litters for evaluation was 22, 22, 21 and 22 in the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Other effects:

no effects observed

Description (incidence and severity):

No other maternal developmental effects were observed.

Details on maternal toxic effects:

The following figures and table are attached to this robust study summary that were discussed and summarized above:
Figure 1: Summary of Group Mean Body Weights: Gestation
Figure 2: Summary of Group Mean Food Consumption: Gestation
Table 1: Summary of Clinical Observations: Gestation
Table 2: Summary of Body Weights: Gestation
Table 3: Summary of Body Weight Gains (g): Gestation
Table 4: Summary of Gravid Uterine Weights and Corrected Body Weights: Gestation
Table 5: Summary of Food Consumption: Gestation
Table 6: Summary of Thyroid Hormone Values
Table 7: Summary of Thyroid Weights
Table 8: Summary of Macroscopic Pathology
Table 9: Summary of Microscopic Pathology
Table 10: Summary of Maternal Performance and Mortality
SEE ATTACHMENT - CONFIDENTIAL!

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean fetal body weights (male, female and combined) were considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size was considered unaffected by treatment with the test item up to 1000 mg/kg/day.
Mean litter sizes were 11.4, 12.0, 10.9 and 10.1 live fetuses/litter in the control, 100, 300 and 1000 mg/kg/day groups, respectively. There were no dead fetuses in this study. The slightly lower mean litter size for individual females in the mid and high dose groups was related to a slightly lower number of corpora lutea and implantation sites. As the treatment with test item is started after implantation (Day 6 post-coitum), this was considered not related to treatment with the test item. The mean litter size was considered to reflect normal biological variation.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female fetuses was considered not to be affected by treatment with the test item up to 1000 mg/kg/day.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In this study, one external malformation was observed in one fetus of the 1000 mg/kg/day group, presenting with polydactyly. Due to its isolated occurrence, this was considered a chance finding.
No external variations were observed in any fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations occurred in 0 (0), 1 (1), 2 (2) and 1 (1) fetuses (litters) in 0, 100, 300 and 1000 mg/kg/day groups, respectively. Two fetuses presented with sternoschisis, one in the low- and one in the mid-dose group. The single occurrences were considered chance findings and not indicative of a test item-related effect. The bent humerus identified in a mid-dose fetus was discovered in isolation and also considered a chance finding. Lastly, the supernumerary phalange was consistent with the external observation of polydactyly in the same fetus, already discussed above (see section 7.4.1). Due to its isolated occurrence, this
was considered a chance finding.
All skeletal variations occurred in the absence of a dose-related incidence trend and/or infrequently. Therefore, they were considered not to be test item-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two visceral malformations were discovered: one fetus in the 300 and 1000 mg/kg/day group each were found to have a small eye, during cephalic examination. Single occurrence at each dose level is not indicative of a test item-related effect and therefore, the small eyes were considered chance findings.
Visceral variations observed in this study affected the liver (supernumerary lobes) and ureter (convoluted and dilated). In the case of liver findings, left lateral supernumerary lobe was only noted in the control group, and left medial and right medial supernumerary lobes in the test item groups were not found to have significantly deviated from the control group and as such did not indicate any test item-relationship. Findings regarding the ureter were incidental and therefore also ruled out as a test item-related effect.

Details on embryotoxic / teratogenic effects:

The following figures and table are attached to this robust study summary that were discussed and summarized above:
Table 11: Summary of Ovarian and Uterine Examinations and Litter Observations
Table 12. Summary of Fetal Abnormalities by Finding
Table 13: Summary of Fetal Abnormalities by Classification
SEE ATTACHMENT - CONFIDENTIAL!

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Table: Summary of Clinical Observations: Gestation

Sex: Females from Day 2 to 21 Observation Type: All Types	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Salivation   Number of Animals Affected   Number of Times Recorded   % of Affected Animals   First to Last seen
	0	1	7	21
	0	2	11	53
	0	5	32	95
	-	10 - 11	8 - 12	7 - 12
Breathing, Abnormal Sounds   Number of Animals Affected   Number of Times Recorded   % of Affected Animals   First to Last seen
	0	0	0	2
	0	0	0	3
	0	0	0	9
	-	-	-	7 - 21
Fur, Erected   Number of Animals Affected   Number of Times Recorded   % of Affected Animals   First to Last seen
	2	1	1	3
	11	3	7	20
	9	5	5	14
	14 - 20	18 - 20	10 - 16	10 - 20
Skin, Scab, Generalized   Number of Animals Affected   Number of Times Recorded   % of Affected Animals   First to Last seen
	0	0	1	0
	0	0	1	0
	0	0	5	0
	-	-	21 - 21	-
Skin, Scab, Ventral Cervical   Number of Animals Affected   Number of Times Recorded   % of Affected Animals   First to Last seen
	0	0	0	2
	0	0	0	10
	0	0	0	9
	-	-	-	11 - 17

 

 

Table: Summary of Body Weights: Gestation / Bodyweight (g)

Sex: Female		Day(s) Relative to Mating (Litter: A)
		2	6	9	12	15	18	21
0 mg/kg/day Group 1	Mean	207.5	219.0	228.3	245.0	256.9	286.6	321.4
	SD	12.4	12.5	13.2	14.4	15.8	18.5	20.0
	N	22	22	22	22	22	22	22
100 mg/kg/day Group 2	Mean	211.5	224.9	235.1	251.3	264.5	294.3	331.5
	SD	13.5	13.8	13.8	14.9	16.5	18.2	22.9
	N	22	22	22	22	22	22	22
	%Diff	1.9	2.7	3.0	2.6	3.0	2.7	3.1
300 mg/kg/day Group 3	Mean	203.6	216.3	226.4	242.8	256.4	284.0	317.2
	SD	12.9	13.6	14.1	14.7	16.8	20.8	24.6
	N	21	21	21	21	21	21	21
	%Diff	-1.9	-1.2	-0.8	-0.9	-0.2	-0.9	-1.3
1000 mg/kg/day Group 4	Mean	206.9	219.5	227.5	241.3	255.0	282.7	307.5
	SD	14.9	16.2	16.7	19.2	20.4	24.6	30.5
	N	22	22	22	22	22	22	22
	%Diff	-0.3	0.2	-0.3	-1.5	-0.7	-1.4	-4.3

 

Table: Summary of Body Weight Gains (g): Gestation / Bodyweight Gain (Interval)

 

Sex: Female		Day(s) Relative to Mating (Litter: A)
		6 → 9 [G]	9 → 12 [G]	12 → 15 [G]	15 → 18 [G]	18 → 21 [G1]	6 → 21 [G]
0 mg/kg/day Group 1	Mean	9.3	16.8	11.9	29.7	34.8	102.4
	SD	4.0	3.6	5.3	5.9	5.7	11.6
	N	22	22	22	22	22	22
100 mg/kg/day Group 2	Mean	10.3	16.2	13.2	29.7	37.2	106.6
	SD	4.4	3.6	5.9	3.8	7.5	13.1
	N	22	22	22	22	22	22
300 mg/kg/day Group 3	Mean	10.1	16.3	13.7	27.6	33.1	100.9
	SD	3.8	2.9	4.4	5.3	8.4	15.3
	N	21	21	21	21	21	21
1000 mg/kg/day Group 4	Mean	8.0	13.8*	13.8	27.6	24.8	88.0**
	SD	3.0	3.8	4.0	7.4	13.5	17.5
	N	22	22	22	22	22	22

[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

[G1] - Kruskal-Wallis & Dunn

 

 

Table: Summary of Gravid Uterine Weights and Corrected Body Weights: Gestation

Sex: Female Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Bodyweight on Day 6 (g) [G]	Mean	219.0	224.9	216.3	219.5
	SD	12.5	13.8	13.6	16.2
	N	22	22	21	22
	%Diff	-	2.7	-1.2	0.2
Terminal Body Weight (g) [G]	Mean	321.4	331.5	317.2	307.5
	SD	20.0	22.9	24.6	30.5
	N	22	22	21	22
	%Diff	-	3.1	-1.3	-4.3
Gravid Uterus Weight (g) [G]	Mean	77.27	82.19	74.78	68.69
	SD	12.21	11.95	16.12	18.40
	N	22	22	21	22
	%Diff	-	6.36	-3.22	-11.11
Adjusted BWG (6-abw) (g) [G]	Mean	25.14	24.45	26.12	19.26
	SD	8.39	7.08	7.30	10.82
	N	22	22	21	22
	%Diff	-	-2.73	3.93	-23.36

 

 

Table: Summary of Food Consumption: Gestation / Food Mean Daily Consumption (g/animal/day)

 

Sex: Female		Day(s) Relative to Mating (Litter: A)
		6 → 9	9 → 12	12 → 15	15 → 18	18 → 21	6 → 21
0 mg/kg/day Group 1	Mean	18.58	19.50	20.11	20.82	19.21	19.64
	SD	2.55	2.23	1.94	2.28	3.68	1.96
	N	22	22	22	22	22	22
100 mg/kg/day Group 2	Mean	19.76	20.42	20.95	20.80	19.94	20.38
	SD	2.25	2.51	2.36	2.56	2.89	1.94
	N	22	22	22	22	22	22
	%Diff	6.36	4.74	4.22	-0.07	3.79	3.73
300 mg/kg/day Group 3	Mean	18.92	19.14	20.49	20.30	18.59	19.49
	SD	2.01	1.67	1.50	2.09	2.42	1.31
	N	21	21	21	21	21	21
	%Diff	1.86	-1.83	1.92	-2.48	-3.25	-0.78
1000 mg/kg/day Group 4	Mean	17.88	17.74*	20.45	20.67	17.85	18.92
	SD	2.07	2.60	1.71	2.29	3.25	1.76
	N	22	22	22	22	22	22
	%Diff	-3.75	-9.01	1.73	-0.73	-7.10	-3.69

Anova & Dunnett: * = p ≤ 0.05

 

Table: Summary of Thyroid Hormone Values / Day: 21 Relative to Mating (Litter: A)

Sex: Female		Reporting Special Chemistry
		T3 (ng/mL) [G]	T4 (ng/mL) [G]	TSH (mU/L) [G]
0 mg/kg/day Group 1	Mean	0.406	21.55	0.3086
	SD	0.088	4.24	0.1713
	N	22	22	22
100 mg/kg/day Group 2	Mean	0.380	19.75	0.2790
	SD	0.079	4.94	0.1362
	N	22	22	22
	tCtrl	0.94	0.92	0.90
300 mg/kg/day Group 3	Mean	0.361	21.42	0.2735
	SD	0.092	5.76	0.1376
	N	21	21	21
	tCtrl	0.89	0.99	0.89
1000 mg/kg/day Group 4	Mean	0.324**	21.92	0.2978
	SD	0.075	4.64	0.1734
	N	22	22	22
	tCtrl	0.80	1.02	0.96

[G] - Anova & Dunnett: ** = p ≤ 0.01

 

Table: Summary of Thyroid Weights

 

Sex: Female Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Terminal Body Weight (g) [G]	Mean	321.4	331.5	317.2	307.5
	SD	20.0	22.9	24.6	30.5
	N	22	22	21	22
	%Diff	-	3.1	-1.3	-4.3
Gland, Thyroid Weight (g) [G1]	Mean	0.01329	0.01423	0.01398	0.01367
	SD	0.00277	0.00216	0.00291	0.00240
	N	22	22	20	22
	%Diff	-	7.11598	5.18303	2.87376
Gland, Thyroid (%bw) [G1]	Mean	0.00412	0.00430	0.00443	0.00453
	SD	0.00076	0.00061	0.00099	0.00116
	N	22	22	20	22
	%Diff	-	4.26134	7.35242	9.84167

[G] - Anova & Dunnett

[G1] - Anova & Dunnett

 

Table: Summary of Macroscopic Pathology

Removal Reason(s): TERMINAL EUTHANASIA Summary: Incidence	Female
	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Animals:	22	22	22	22
GLAND, THYROID   Submitted   No Visible Lesions   Small
	22	22	22	22
	21	22	22	22
	1	0	0	0
LIVER   Submitted   Abnormal appearance; lateral lobe
	1	0	0	0
	1	.	.	.
PLACENTA   Submitted   Abnormal appearance
	1	0	0	0
	1	.	.	.
SKIN   Submitted   Scab; flank
	0	0	1	0
	.	.	1	.

 

Table: Summary of Microscopic Pathology

Removal Reason(s): TERMINAL EUTHANASIA Summary: Incidence	Female
	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Animals:	22	22	22	22
GLAND, THYROID   Examined   No Visible Lesions   Ectopia; thymic   .... minimal   Hypertrophy; follicular cell   .... minimal
	22	22	22	22
	19	21	20	20
	2	0	0	0
	2	0	0	0
	1	1	2	2
	1	1	2	2

 

Table: Summary of Maternal Performance and Mortality

Sex: Female Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Group Size - Females		22	22	22	22
Number of Females Pregnant [f]	N+ve	22	22	21	22
	%	100.0	100.0	95.5	100.0
Female with Live Fetuses [f]	N+ve	22	22	21	22
	%	100.0	100.0	100.0	100.0
Total Resorptions [f] N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Female with all Nonviable [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Terminal Euthanasia [f]	N+ve	22	22	22	22
	%	100.0	100.0	100.0	100.0
Unscheduled Death/Euthanasia [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0

[f] - Fisher's Exact

 

Table: Summary of Ovarian and Uterine Examinations and Litter Observations

Sex: Female Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Female with Live Fetuses	N+ve	22	22	21	22
	%	100.0	100.0	100.0	100.0
Number of Corpora Lutea [k]	Mean	13.1	13.1	12.4	12.3
	SD	1.3	1.9	1.6	2.1
	N	22	22	21	21
	%Diff	-	0.0	-5.4	-6.2
Number of Implantations [k]	Mean	11.9	12.4	11.5	10.9
	SD	1.6	1.8	2.1	2.5
	N	22	22	21	22
	%Diff	-	4.6	-2.9	-8.4
Pre-implantation Loss (%) [k]	Mean	8.93	4.99	7.17	10.99
	SD	12.08	5.56	11.99	15.30
	N	22	22	21	21
	%Diff	-	-44.08	-19.65	23.05
Total Number of Fetuses [k]	Mean	11.4	12.0	10.9	10.1
	SD	1.8	1.9	2.4	3.0
	N	22	22	21	22
	%Diff	-	5.6	-4.8	-11.2
Number of Live Fetuses [k]	Mean	11.4	12.0	10.9	10.1
	SD	1.8	1.9	2.4	3.0
	N	22	22	21	22
	%Diff	-	5.6	-4.8	-11.2
Number of Dead Fetuses [k]	Mean	0.0	0.0	0.0	0.0
	SD	0.0	0.0	0.0	0.0
	N	22	22	21	22
	%Diff	-	-	-	-
Number of Early Resorptions [k]	Mean	0.5	0.3	0.7	0.7
	SD	0.7	0.6	0.9	1.0
	N	22	22	21	22
	%Diff	-	-30.0	46.7	60.0
Number of Late Resorptions [k]	Mean	0.0	0.0	0.0	0.0
	SD	0.0	0.2	0.0	0.0
	N	22	22	21	22
	%Diff	-	-	-	-
Total Number of Resorptions [k]	Mean	0.5	0.4	0.7	0.7
	SD	0.7	0.6	0.9	1.0
	N	22	22	21	22
	%Diff	-	-20.0	46.7	60.0
Post-implantation Loss (%) [k]	Mean	3.85	2.98	6.33	8.23
	SD	5.63	4.80	8.92	14.13
	N	22	22	21	22
	%Diff	-	-22.60	64.35	113.68
Number of Live Male Fetuses [k]	Mean	5.8	6.3	5.3	4.8
	SD	1.8	2.1	1.9	2.0
	N	22	22	21	22
	%Diff	-	8.6	-8.3	-17.2
Number of Live Female Fetuses [k]	Mean	5.6	5.7	5.5	5.3
	SD	1.7	2.2	1.7	2.4
	N	22	22	21	22
	%Diff	-	2.4	-1.2	-4.9
Live Male Fetus/Litter (%) [k]	Mean	50.72	52.77	48.96	47.78
	SD	12.69	15.36	11.64	16.06
	N	22	22	21	22
	%Diff	-	4.04	-3.47	-5.80
Live Female Fetuses/Litter (%) [k]	Mean	49.28	47.23	51.04	52.22
	SD	12.69	15.36	11.64	16.06
	N	22	22	21	22
	%Diff	-	-4.15	3.57	5.97
Mean Fetal Weight males (g) [G]	Mean	5.144	5.140	5.206	5.074
	SD	0.281	0.210	0.281	0.378
	N	22	22	21	22
	%Diff	-	-0.071	1.223	-1.361
Mean Fetal Weight females (g) [G]	Mean	4.848	4.861	4.968	4.830
	SD	0.275	0.233	0.242	0.359
	N	22	22	21	22
	%Diff	-	0.279	2.480	-0.377
Mean Fetal Weight all (g) [G]	Mean	4.991	5.012	5.078	4.949
	SD	0.235	0.202	0.232	0.347
	N	22	22	21	22
	%Diff	-	0.423	1.748	-0.832
Mean Fetal AGD males (mm) [G1]	Mean	2.84	2.81	2.85	2.76
	SD	0.17	0.20	0.18	0.18
	N	22	22	21	22
	%Diff	-	-1.13	0.39	-2.58
Mean Fetal AGD females (mm) [G1]	Mean	1.40	1.44	1.42	1.37
	SD	0.22	0.23	0.20	0.19
	N	22	22	21	22
	%Diff	-	2.69	1.94	-1.95
Mean Normalized Fetal AGD m [G]	Mean	1.646	1.627	1.645	1.611
	SD	0.115	0.106	0.100	0.101
	N	22	22	21	22
	%Diff	-	-1.175	-0.075	-2.154
Mean Normalized Fetal AGD f [G]	Mean	0.827	0.848	0.836	0.811
	SD	0.132	0.132	0.120	0.103
	N	22	22	21	22
	%Diff	-	2.474	0.998	-2.007

[k] - Kruskal-Wallis & Dunn

[G] - Anova & Dunnett

[G1] - Anova & Dunnett

 

Tables: Summary of Fetal Abnormalities by Finding

Exam Type: External		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:		251	265	228	223
Number of Fetuses Evaluated:		251	266	228	223
Number of Litters Examined:		22	22	21	22
Number of Litters Evaluated:		22	22	21	22
Paw/digit
Hindpaw, Left, Polydactyly – Malformation	Fetuses N(%)	0(0.00)	0(0.00)	0(0.00)	1(0.41)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(4.5)

[Fetuses %] - Kruskal-Wallis & Dunn

FetusesN(%) N=Group Fetal Incidence;(%)=Mean Litter % of Fetuses with the Abnormality

 

		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:		128	132	114	109
Number of Fetuses Evaluated:		251	266	228	223
Number of Litters Examined:		22	22	21	22
Number of Litters Evaluated:		22	22	21	22
Exam Type: Fixed Head
Eye
Eye, Left, Small – Malformation	Fetuses N(%)	0(0.00)	0(0.00)	1(0.68)	1(0.91)
	Litters N(%)	0(0.0)	0(0.0)	1(4.8)	1(4.5)
Exam Type: FreshVisBody
Liver
Lobe, Left lateral, Supernumerary - Variation	Fetuses N(%)	1(0.76)	0(0.00)	0(0.00)	0(0.00)
	Litters N(%)	1(4.5)	0(0.0)	0(0.0)	0(0.0)
Lobe, Left medial, Supernumerary - Variation	Fetuses N(%)	4(3.33)	13(10.84)	5(3.74)	4(3.33)
	Litters N(%)	2(9.1)	7(31.8)	3(14.3)	3(13.6)
Lobe, Right medial, Supernumerary - Variation	Fetuses N(%)	2(1.67)	7(5.05)	4(6.51)	9(9.18)
	Litters N(%)	2(9.1)	7(31.8)	3(14.3)	6(27.3)
Ureter
Ureter, Left, Convoluted - Variation	Fetuses N(%)	0(0.00)	1(0.76)	1(0.68)	0(0.00)
	Litters N(%)	0(0.0)	1(4.5)	1(4.8)	0(0.0)
Ureter, Left, Dilatation, Minimal - Variation	Fetuses N(%)	0(0.00)	0(0.00)	1(0.68)	1(0.65)
	Litters N(%)	0(0.0)	0(0.0)	1(4.8)	1(4.5)
Ureter, Right, Convoluted, Minimal - Variation	Fetuses N(%)	0(0.00)	0(0.00)	1(0.95)	1(2.27)
	Litters N(%)	0(0.0)	0(0.0)	1(4.8)	1(4.5)

 

 

		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:		123	133	114	114
Number of Fetuses Evaluated:		251	266	228	223
Number of Litters Examined:		22	22	21	22
Number of Litters Evaluated:		22	22	21	22
Exam Type: Skeletal
Forelimb
Humerus, Right, Bent - Malformation	Fetuses N(%)	0(0.00)	0(0.00)	1(2.38)	0(0.00)
	Litters N(%)	0(0.0)	0(0.0)	1(4.8)	0(0.0)
Hindlimb
Hindpaw phalanges, 1 or more, Supernumerary - Malformation	Fetuses N(%)	0(0.00)	0(0.00)	0(0.00)	1(0.76)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(4.5)
Pelvic girdle
Ilium, Both, Misaligned – Variation	Fetuses N(%)	5(4.85)	3(2.27)	5(5.71)	7(5.42)
	Litters N(%)	4(18.2)	3(13.6)	5(23.8)	6(27.3)
Rib
Costal cartilage, 1 or more, Fused - Variation	Fetuses N(%)	0(0.00)	0(0.00)	0(0.00)	1(0.91)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(4.5)
Rib, 1 or more, Wavy rib – Variation	Fetuses N(%)	32(26.89)	43(33.61)	25(24.17)	34(28.74)
	Litters N(%)	16(72.7)	16(72.7)	12(57.1)	14(63.6)
Scapula
Scapula, Both, Bent – Variation	Fetuses N(%)	1(0.76)	0(0.00)	1(0.95)	0(0.00)
	Litters N(%)	1(4.5)	0(0.0)	1(4.8)	0(0.0)
Scapula, Right, Bent – Variation	Fetuses N(%)	0(0.00)	2(1.48)	2(3.17)	0(0.00)
	Litters N(%)	0(0.0)	2(9.1)	2(9.5)	0(0.0)
Skull
Frontal, Both, Incomplete ossification – Variation	Fetuses N(%)	0(0.00)	0(0.00)	2(1.59)	3(2.58)
	Litters N(%)	0(0.0)	0(0.0)	1(4.8)	3(13.6)
Exam Type: Skeletal
Interparietal, Incomplete ossification - Variation	Fetuses N(%)	24(18.27)	23(17.65)	13(12.78)	18(15.91)
	Litters N(%)	11(50.0)	12(54.5)	7(33.3)	9(40.9)
Parietal, Both, Incomplete ossification - Variation	Fetuses N(%)	10(7.60)	10(8.15)	12(10.12)	13(11.17)
	Litters N(%)	7(31.8)	9(40.9)	7(33.3)	6(27.3)
Parietal, Left, Incomplete ossification - Variation	Fetuses N(%)	0(0.00)	1(0.76)	0(0.00)	1(0.91)
	Litters N(%)	0(0.0)	1(4.5)	0(0.0)	1(4.5)
Parietal, Right, Incomplete ossification - Variation	Fetuses N(%)	8(6.19)	4(3.41)	1(0.95)	2(1.67)
	Litters N(%)	6(27.3)	4(18.2)	1(4.8)	2(9.1)
Squamosal, Both, Incomplete ossification - Variation	Fetuses N(%)	4(2.81)	4(3.41)	6(5.16)	4(3.30)
	Litters N(%)	4(18.2)	4(18.2)	2(9.5)	4(18.2)
Squamosal, Left, Incomplete ossification	- Variation	Fetuses N(%)	1(0.91)	1(1.14)	0(0.00)
	Litters N(%)	1(4.5)	1(4.5)	0(0.0)	3(13.6)
Squamosal, Right, Incomplete ossification - Variation	Fetuses N(%)	3(2.58)	2(1.52)	1(0.79)	1(0.76)
	Litters N(%)	3(13.6)	2(9.1)	1(4.8)	1(4.5)
Supraoccipital, Incomplete ossification – Variation	Fetuses N(%)	8(6.60)	6(4.89)	5(4.37)	7(6.06)
	Litters N(%)	6(27.3)	6(27.3)	3(14.3)	5(22.7)
Zygomatic arch, Both, Incomplete ossification - Variation	Fetuses N(%)	1(0.91)	3(2.80)	6(4.65)	5(4.55)
	Litters N(%)	1(4.5)	3(13.6)	2(9.5)	3(13.6)
Zygomatic arch, Left, Incomplete ossification - Variation	Fetuses N(%)	2(1.56)	2(1.67)	2(1.59)	1(0.91)
	Litters N(%)	2(9.1)	2(9.1)	2(9.5)	1(4.5)
Zygomatic arch, Right, Incomplete ossification - Variation	Fetuses N(%)	7(5.54)	2(1.33)	2(1.75)	2(1.82)
	Litters N(%)	7(31.8)	2(9.1)	2(9.5)	2(9.1)
Sternebra
Sternebra, 1 or more, Branched - Variation	Fetuses N(%)	1(0.76)	2(1.41)	0(0.00)	0(0.00)
	Litters N(%)	1(4.5)	2(9.1)	0(0.0)	0(0.0)
Sternebra, 1 or more, Misaligned - Variation	Fetuses N(%)	3(2.16)	1(0.76)	6(5.09)	3(3.79)
	Litters N(%)	3(13.6)	1(4.5)	5(23.8)	3(13.6)
Sternebra, 1 or more, Sternoschisis - Malformation	Fetuses N(%)	0(0.00)	1(0.76)	1(0.79)	0(0.00)
	Litters N(%)	0(0.0)	1(4.5)	1(4.8)	0(0.0)
Sternebra, 1 or more, Unossified - Variation	Fetuses N(%)	0(0.00)	0(0.00)	0(0.00)	1(2.27)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(4.5)
Sternebra, 1 or more, Wide - Variation	Fetuses N(%)	1(0.76)	1(0.76)	0(0.00)	0(0.00)
	Litters N(%)	1(4.5)	1(4.5)	0(0.0)	0(0.0)
Sternebra, 1 or more, Incomplete ossification – Variation	Fetuses N(%)	1(0.65)	0(0.00)	1(1.19)	2(2.84)
	Litters N(%)	1(4.5)	0(0.0)	1(4.8)	2(9.1)
Supernumerary rib
Cervical, 1 or more, Full - Variation	Fetuses N(%)	4(3.38)	0(0.00)	1(1.19)	1(0.91)
	Litters N(%)	3(13.6)	0(0.0)	1(4.8)	1(4.5)
Cervical, 1 or more, Short - Variation	Fetuses N(%)	5(4.13)	3(2.42)	6(5.16)	6(6.36)
	Litters N(%)	3(13.6)	3(13.6)	4(19.0)	5(22.7)
Thoracolumbar, 1 or more, Full - Variation	Fetuses N(%)	6(4.70)	9(6.63)	9(8.02)	12(9.19)
	Litters N(%)	5(22.7)	5(22.7)	8(38.1)	7(31.8)
Thoracolumbar, 1 or more, Short - Variation	Fetuses N(%)	74(60.24)	78(59.12)	61(52.53)	69(59.68)
	Litters N(%)	21(95.5)	21(95.5)	19(90.5)	20(90.9)
Vertebra
Thoracic centrum, 1 or more, Incomplete ossification - Variation	Fetuses N(%)	2(1.67)	5(4.03)	2(1.98)	1(0.76)
	Litters N(%)	2(9.1)	2(9.1)	2(9.5)	1(4.5)

[Fetuses %] - Kruskal-Wallis & Dunn

FetusesN(%) N=Group Fetal Incidence;(%)=Mean Litter % of Fetuses with the Abnormality

 

Tables: Summary of Fetal Abnormalities by Classification

	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:	251	265	228	223
Number of Fetuses Evaluated:	251	266	228	223
Number of Litters Examined:	22	22	21	22
Number of Litters Evaluated:	22	22	21	22
Exam Type: External
Malformation
Number of Fetuses	0	0	0	1
Litter % of Fetuses [k]	0.00	0.00	0.00	0.41
Number of Litters	0	0	0	1
All classifications
Number of Fetuses	0	0	0	1
Litter % of Fetuses [k]	0.00	0.00	0.00	0.41
Number of Litters	0	0	0	1

 

	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:	128	132	114	109
Number of Fetuses Evaluated:	251	266	228	223
Number of Litters Examined:	22	22	21	22
Number of Litters Evaluated:	22	22	21	22
Exam Type: Fixed Head
Malformation
Number of Fetuses	0	0	1	1
Litter % of Fetuses [k]	0.00	0.00	0.68	0.91
Number of Litters	0	0	1	1
All classifications
Number of Fetuses	0	0	1	1
Litter % of Fetuses [k]	0.00	0.00	0.68	0.91
Number of Litters	0	0	1	1

[k] - Kruskal-Wallis & Dunn

 

	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:	128	132	114	109
Number of Fetuses Evaluated:	251	266	228	223
Number of Litters Examined:	22	22	21	22
Number of Litters Evaluated:	22	22	21	22
Exam Type: FreshVisBody
Variation
Number of Fetuses	7	20	10	15
Litter % of Fetuses [k]	5.76	15.74	11.20	15.43
Number of Litters	3	12	7	10
All classifications
Number of Fetuses	7	20	10	15
Litter % of Fetuses [k]	5.76	15.74	11.20	15.43
Number of Litters	3	12	7	10

[k] - Kruskal-Wallis & Dunn

 

	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:	123	133	114	114
Number of Fetuses Evaluated:	251	266	228	223
Number of Litters Examined:	22	22	21	22
Number of Litters Evaluated:	22	22	21	22
Exam Type: Skeletal
Variation
Number of Fetuses	102	109	83	91
Litter % of Fetuses [k]	82.53	82.72	73.03	78.83
Number of Litters	22	22	20	21
Malformation
Number of Fetuses	0	1	2	1
Litter % of Fetuses [k]	0.00	0.76	3.17	0.76
Number of Litters	0	1	2	1
All classifications
Number of Fetuses	102	109	83	91
Litter % of Fetuses [k]	82.53	82.72	73.03	78.83
Number of Litters	22	22	20	21

[k] - Kruskal-Wallis & Dunn

Conclusions:

In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Maleated TOFA were established as being at least 1000 mg/kg/day.

Executive summary:

The objectives of this study were to determine the potential of Maleated TOFA to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Days 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

Time-mated female Wistar Han rats were treated with Maleated TOFA from Days 6 to 20 post-coitum, inclusive by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day. The rats of the control group received the vehicle, propylene glycol. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

No maternal toxicity was observed up to the highest dose level tested (1000 mg/kg/day) and all animals survived until scheduled necropsy.

At 1000 mg/kg/day, a minor decrease in mean food consumption between Days 9-12 and 18-21 post-coitum, and consequently a lower mean body weight and body weight gain was noted. Based on the small effect, this decrease was considered to be not adverse.

A decrease in corrected body weight gain and gravid uterus was noted at 1000 mg/kg/day. Based on the contribution of a few individual females and occurrence in the control group, this effect was considered not adverse at the magnitude of change observed.

Mean serum levels of total triiodothyronine (T3) showed an apparent dose-related trend towards a decrease across the dose groups, with statistical significance at 1000 mg/kg/day. As mean total T3 remained within the historical control data range at these dose levels, this decrease was considered not to represent an adverse effect.

No test item-related or toxicologically significant changes were noted in any of the remaining maternal parameters investigated in this study (i.e. clinical appearance, thyroid hormone levels (thyroxine (T4), thyroid stimulating hormone (TSH)), thyroid gland weights, macroscopic evaluation, microscopic evaluation of the thyroid gland, uterine contents, corpora lutea, implantation sites and pre- and post-implantation loss).

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).

In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Maleated TOFA were established as being at least 1000 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

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:

other: OECD 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (2016)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

HD0379UD11

Species:

rat

Strain:

Wistar

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 males were approximately 11 weeks old and 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

Vehicle:

polyethylene glycol

Details on 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 %.

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.

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

Duration of test:

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.

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.

Maternal examinations:

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

Ovaries and uterine content:

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.

Fetal examinations:

Any foetuses were examined externally where possible and discarded.

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):

Females: Mean body weight gain in all treated groups was higher compared with the control during the premating period.

Food efficiency:

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:

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.

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

Number of abortions:

no effects observed

Description (incidence and severity):

There was no test item-related effect on the percentage pre-birth loss in any group.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was a slightly lower mean number of implantation sites in the 400 mg/kg/day (10.9) compared with the concurrent control (12.3). However, the mean value was disproportionately lowered by three females with a number of implantation sites ≤ 8 compared with 2, 1 and none in the 200, 100 mg/kg/day and control groups, respectively. However, since the mean value in the high dose group remained within the historical control data range (10.2 to 15.0) this was considered to be no toxicological significance.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean duration of gestation was comparable (approximately 22 days) in all groups.

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical biochemistry

clinical signs

dead fetuses

effects on pregnancy duration

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

urinalysis

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no test item related effect on pup viability in any group.

Changes in sex ratio:

not examined

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no test item related effect on mean live litter size or pup viability in any group.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Under the experimental conditions of this OECD 422 study, the daily oral (gavage) administration of the test item 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:

In this OECD 422 study there were 9/10, 9/10, 7/10 and 9/10 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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the absence of fertility and developmental effects in an OECD 422 study, dosed up to 400 mg/kg bw/d, and absence of developmental effects in an OECD 414 study, no classification for reproduction toxicity is required in line with CLP (Regulation EC No. 1272/2008).